Controllable transcription

Abstract

To provide a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within the inducible cassette.SOLUTION: The method may be used for any cell type from any eukaryotic organism, but has a particular application in the introduction of the inducible cassette into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted to ensure that the genetic material it contains is neither silenced nor subject to negative influences from the insertion site, and that transcription of the genetic material is controlled.SELECTED DRAWING: Figure 4a-c

Description

Technical Field

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

Background Art

[0002] (Background of the Invention) Stem cell research holds great promise for the study of human development, regenerative medicine, disease modeling, drug discovery, and cell transplantation. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult. In addition, stem cell-derived cells enable the study of the physiological and pathological responses of human cell populations that are not easily accessible. This often involves the study of genes (and other forms of regulatory mechanisms encoded within non-protein-coding RNA - ncRNA). Unfortunately, controllable transcription or expression of genetic information in human cells has proven to be particularly difficult.

[0003] For several important aspects of regenerative medicine, disease modeling, drug discovery, and cell transplantation, the manipulation and production of mature human cell types from easily accessible sources are required. The control of transgene expression in human cells is fundamental to biological research. However, this has proven to be difficult in human cells. Also, suitable for the purposes of drug discovery and regenerative medicine For several important aspects of regenerative medicine, disease modeling, drug discovery, and cell transplantation, the manipulation and production of mature human cell types from easily accessible sources are required. The control of transgene expression in human cells is fundamental to biological research. However, this has proven to be difficult in human cells. Also, suitable for the purposes of drug discovery and regenerative medicine For several important aspects of regenerative medicine, disease modeling, drug discovery, and cell transplantation, the manipulation and production of mature human cell types from easily accessible sources are required. The control of transgene expression in human cells is fundamental to biological research. However, this has proven to be difficult in human cells. Also, suitable for the purposes of drug discovery and regenerative medicine In terms of quantity and quality, in vitro derivation of many highly desirable human cell types is actually needed. This is because directed differentiation of stem cells into the desired cell type is often difficult. Other approaches have emerged, including direct reprogramming of cells into different types. In particular, multi-type cells including hPSCs. Forward programming is a method for directly converting potent stem cells into mature cell types, in humans. This is recognized as a powerful strategy for inducing cells. This reprogramming is for stem cells. To convert to a specific mature cell type, key lineage transcription factors (or IncRNA and micro It involves the forced expression of non-coding RNA (including RNA). Also, due to this, in human cells Controllable expression of genetic information is being challenged. Currently, available forward programming The protocol is primarily based on lentiviral transduction of cells, randomly This results in diverse expression or complete silencing of the inserted induction cassette. This necessitates an additional purification step to isolate subpopulations that express key transcription factors. Therefore, further improvements to these methods are clearly needed.

[0004] In addition to the induction of transgene expression, in order to enable loss-of-function studies, cells The ability to control the knockdown and knockout of genes or other coding sequences within the body is extremely important. This is desirable. Research into loss of function in stem cells and mature cell types is important for human development, disease and life It provides a unique opportunity to study the mechanisms that regulate this principle. However, current technology This does not enable the easy and efficient manipulation of gene expression. To induce gene knockdown... Current techniques for introducing substances such as inducible small hairpin RNA (shRNA) into stem cells The procedure involves the silencing of the introduced gene and the position-effect limiting activity, as described above, and the forward regeneration. We are plagued by many shortcomings in programming. Therefore, the function in stem cells Inducible gene knockout and knockdown in stem cells enable loss research. It is needed.

[0005] Any of the above improvements to the method may have other negative effects related to silencing and integration. Stable transcription of genetic material contained within induction cassettes such as transgenes that are resistant to It must be ensured that this is achieved. Silencing involves DNA methylation or hi It can be caused by multiple epigenetic mechanisms, including stone modification. Using conventional techniques based on viral transduction, the cells obtained have completely transgenes. These are heterogeneous populations that either express, partially express, or silence the gene. This is undesirable for many applications. Viral vectors can transfer their genetic material. It is incorporated into the transcriptional activity region of Nomu, thereby increasing the likelihood of oncogenic events caused by insertional mutagenesis. It shows a tendency to...

[0006] For many applications, the induction cassette switches on as needed, and high levels To control the transcription of genetic material inserted into cells so that it can be transcribed at a specific level, including It is desirable that the insertion of the induction cassette be random within the genome. It cannot be achieved.

[0007] Therefore, the present inventors have made it possible to stably introduce induction cassettes into the genome of cells. On the other hand, we developed a method that can control the transcription of that induction cassette. This is particularly important. introducing the induction cassette into pluripotent stem cells to control the transcription of the inserted genetic material has advantages for any cell type that is desired. The induction cassette can contain any genetic material capable of being transcribed, such as a transgene or a non-coding RNA (ncRNA). The substance contained within the induction cassette is determined by the effect required from the stem cells, including the expression of the transgene or gene knockdown or knockout. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] (Summary of the Invention) The inventors have found that by using the dual genomic safe harbor targeting system described herein, it is possible to insert an induction cassette and control the transcription of the genetic material therein. The risk of epigenetic silencing of the inserted genetic material is reduced, and it is possible to obtain a homogeneous population of cells that transcribe the induction cassette, so such a method is highly desirable. MEANS FOR SOLVING THE PROBLEM

[0009] Therefore, the present invention is a method for controlling the transcription of a gene sequence in a cell, comprising: a) a step of targetedly inserting a transcription regulatory protein-coding gene into a first gene safe harbor site; and b) a step of targetedly inserting an induction cassette into a second gene safe harbor site, wherein the induction cassette contains the gene sequence operably linked to an inducible promoter, and the promoter is regulated by the transcription regulatory protein, the step, including, ​The present invention relates to the method wherein the first and second gene-safe harbor sites are different.

[0010] The incorporation of induction cassettes to specific genome-safe harbor sites (GSHs) into the genome It is preferable to random insertion. GSH does not adversely affect host cells or organisms. The human genome can respond to the predictable expression of newly incorporated DNA. This was previously defined as an extragenetic region. A useful safe harbor is the desired level of tan. Sufficient inserted genes to obtain a protein (through further translation) or non-coding RNA. The transcription of the sequence must be made possible. GSH also makes cells more susceptible to malignant transformation. This must not be done, and cellular function must not be altered (Sadelain et al., 2012, Nature Reviews Ca). ncer, 12(1), 51-8. doi:10.1038 / nrc3179).

[0011] The first gene-safe harbor site is at least one transcriptionally regulatory protein-coding gene. It is used to introduce a gene. A transcription regulatory protein (or transcription factor) is a gene that modifies the gene. Increases transcription. Most transcription regulators are enhancers that are operably linked to genes. It is a DNA-binding protein that binds to the proximal element of the promoter or similar. [Effects of the Invention]

[0012] In some embodiments, transcription regulatory proteins are constitutively expressed and permanently within cells. It is expressed in [location]. Therefore, transcription regulatory proteins are operably linked to constitutive promoters. It is possible. Constitutive promoters are responsible for most tissues and fine tissues at all stages of growth and development. It directs gene expression uniformly within the cell. The constitutive promoter is used in the method of the present invention. In this case, it confers a high level of gene expression.

[0013] Further genetic material, including genes, can be inserted into the first GSH containing transcriptional regulatory proteins. Such genes, for example, indicate that a transcription regulatory protein has been successfully inserted. It may contain one or more markers, such as green fluorescent protein (GFP), which can be used for this purpose. Other options include genes that enable gene editing, such as Cas9 and its derivatives or CasL. For assaying the endogenous or exogenous expression of specific genes in cells, as well as their derivatives. It includes reporter sequences that can be used for this purpose.

[0014] The second GSH is an induction cassette in which the desired gene sequence is operably linked to an induction promoter. It is used to introduce a promoter. Such promoters are controlled by transcription regulatory proteins. Transcription is only enabled if it is correctly induced. Transcription regulatory proteins are external to the cell. It can be controlled by the substances supplied. Therefore, the presence of exogenous substances can induce promo Expression from a receptor can be enabled or blocked. Examples of such controllable expression include The Tet-ON system is included and will be further described herein.

[0015] Further induction cassettes may be inserted into further GSHs, the GSHs being the above-mentioned ones This is different from GSH 1 and 2.

[0016] One or more gene sequences can be controlledly transcribed from within a second and / or further GSH. The induction cassette is to be inserted into GSH, and its transcription is to be controlled. It may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 gene sequences.

[0017] The gene sequences or multiple gene sequences that are desired to be inserted into GSH or multiple GSHs are induction These gene sequences are operablely linked to the promoter and reside within the induction cassette. Any appropriate method that can be transcribed into RNA after promoter activity has been induced. It can be any sequence. The appropriate gene sequence is the transgene (the RNA produced is a polypeptide). Protein-coding genes (protein-coding RNAs, which are translated messenger RNAs (mRNAs)), non-coding ncRNA (ncRNA - shRNA), antisense RNA (asRNA), guide RNA (gRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting RNA (tasiRNA), antagonist miRNA, app This includes, but is not limited to, tamers, miRNA sponges, and any other functional RNAs. This includes, but is not limited to, these items.

[0018] The induction cassette may contain additional genetic material to be inserted into a second or further GSH. Such additional genetic material is used to indicate that transcription is occurring, and green fluorescent protein (GF) is used to indicate that transcription is taking place. It may include one or more markers such as P). Alternatively, it may also include antibiotic or drug resistance genes. The genes of offspring, for example, can enable the selection of a properly inserted induction cassette. When it is desirable to induce the expression of a specific gene or a sequence that interferes with its function in order to study ability. There are also gene expressions to enhance or inhibit the biological functions of cells, or Gene expression that affects cells in other parts of an organism includes growth factors, insulin, and others. In some cases, this may be desirable, including the expression of peptide hormones.

[0019] Technically, insertions into the first and / or second GSH can occur on one chromosome or on both chromosomes. It is possible. GSH is present at the same locus on both chromosomes in diploid organisms. This allows for increased transcription levels from genetic material inserted into the retrieval, and therefore, especially high levels. Insertion into both chromosomes is advantageous because it allows for Bell's transcription to be achieved.

[0020] Insertion into GSH is a customized site for DNA double-strand breaks (DSBs) that can be achieved in GSH. Based on specific generation, it is possible to control the specific insertion of genetic material into specific GSHs. Next, the genetic material can be introduced using any appropriate mechanism, such as homologous recombination. Any method can be used to create a specific DSB within the system, but the preferred system is This includes CRISPR / Cas9 and its modified versions, ZFN, and TALEN systems.

[0021] Furthermore, the insertion of transcriptional regulators and / or induction cassettes is designed to be reversible. The inserted genetic material can be removed and / or replaced if necessary. It may be replaced with a transcription regulator / induction cassette. Replace the transcription regulator and / or induction cassette. The method constitutes part of the present invention. Such substitution is performed when the cell culture is one transcription regulator. and / or if successfully modified in one induction cassette, and if transcription regulators and / or inductions This may be useful when it is desirable to replace the cassette. This is for already successful insertions. By utilizing this, it may be possible to perform larger insertions. Therefore, the implant allows for the removal of all or part of the implant from the GSH, such as a portion of the implant. It may contain cleaved sequences. Preferred methods of removal or substitution include recombination.

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

[0023] In one embodiment, the present invention is a method for controlling the expression of a transgene in a cell, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A second gene-safe harbor for an introduced gene operably linked to an inductive promoter. A step of targeted insertion into a site, wherein the inducing promoter is a transcription regulatory protein The above process is adjusted as follows: Includes, The present invention provides the method wherein the first and second gene-safe harbor sites are different.

[0024] In this embodiment of the present invention, the aforementioned induction cassette is operably connected to the induction promoter. Includes a linked transgene. In this embodiment of the present invention, desired to be included in the induction cassette The gene sequence is an introduced gene, preferably a protein-coding gene. Therefore, The transcription and translation (expression) of the introduced gene can be controlled within the cell. The advantage of this method is that it is necessary In some cases, this allows for the overexpression of the introduced gene.

[0025] Furthermore, in this embodiment of the present invention, further identical or different transgenes are the first and It can be inserted into a second GSH and a further GSH that is different from the above induction. Such a transgene can be inserted into the induction It is operably connected to the promoter.

[0026] In one embodiment, the present invention relates to a method for controlling the transcription of non-coding RNA in cells. And, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A step of targeted insertion of an induction cassette into a second gene-safe harbor site, the same as above The induction cassette operatively ligates a non-coding RNA sequence to the induction promoter. The DNA comprises encoding DNA, and the promoter is regulated by the transcription regulatory protein. The aforementioned process, Includes, The present invention provides the method wherein the first and second gene-safe harbor sites are different.

[0027] Furthermore, in this embodiment of the present invention, further identical or different induction cassettes are the first and It can be inserted into a second GSH and a further GSH different from the first. Such induction cassettes are non-corrupted. Any other DNA or inductive promoter that encodes a Ding RNA sequence and is operably ligated to it The gene sequence may include the promoter, which is regulated by the transcription regulatory protein. .

[0028] More specifically, this method allows for the knockdown of endogenous genes in cells. Therefore, the present invention relates to a method for reducing the transcription and / or translation of endogenous genes within a cell. There is, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A step of targeted insertion of an induction cassette into a second gene-safe harbor site, the same as above The induction cassette operatively ligates a non-coding RNA sequence to the induction promoter. The promoter contains encoding DNA, and the promoter is regulated by the transcription regulatory protein. The non-coding RNA sequence suppresses the transcription or translation of an endogenous gene in the above step. Includes, The present invention provides the method wherein the first and second gene-safe harbor sites are different.

[0029] Furthermore, in this embodiment of the present invention, further identical or different induction cassettes are the first and It can be inserted into a second GSH and a further GSH different from the first. Such induction cassettes are non-corrupted. Any other DNA or inductive promoter that encodes a Ding RNA sequence and is operably ligated to it The gene sequence may include the promoter, which is regulated by the transcription regulatory protein. .

[0030] In any aspect or embodiment, the endogenous gene is a protein or noncoded It can encode GLRNA.

[0031] In the two embodiments of the present invention described above, the induction cassette(s) are non-coding R It contains DNA that codes for NA, that is, RNA that is functional but not translated into proteins. This non-coding RNA could be any suitable RNA, such as those previously described, Preferably, it is a low molecular weight hairpin RNA (shRNA). In the latter embodiment of the present invention, non Coding RNAs generally inhibit the transcription or translation of genes, or prevent their expression. By doing so, gene knockdown can be achieved in any suitable way. Ultimately, the above Gene expression is reduced or inhibited, but the gene itself remains intact.

[0032] Alternatively, non-coding RNA contained within the sequence of the induction cassette is, in particular, a gene itself. Used to replace or destroy the body, or to knock out endogenous genes within cells. It may contain RNA that can be used in this embodiment of the present invention. Ding RNA is an element of the CRISPR / Cas9 platform, more specifically, an endogenous gene. It contains guide RNA (gRNA) directed to target genes.

[0033] Therefore, in one embodiment, the present invention relates to a method for knocking out endogenous genes within cells. It is a law, a) The first of the transcriptional regulatory protein-coding genes and the genes encoding Cas9 or its derivatives The process of targeted insertion into a gene-safe harbor site; and b) A step of targeted insertion of an induction cassette into a second gene-safe harbor site, the same as above The induction cassette includes a guide RNA operably ligated to the induction promoter, and the promoter The motor is regulated by the transcription regulatory protein, and the gRNA sequence marks an endogenous gene. To target, the above process, Includes, The present invention provides the method wherein the first and second gene-safe harbor sites are different.

[0034] Furthermore, in this embodiment of the present invention, further identical or different induction cassettes are the first and It can be inserted into a second GSH and a further GSH different from the first. Such an induction cassette is an induction pro It may include any gene sequence operably linked to a motor, and the promoter may rotate It is regulated by regulatory proteins.

[0035] Therefore, in the above embodiment of the present invention, gRNA transcription is controlledly induced.

[0036] In a further embodiment, the present invention reduces the transcription and / or translation of endogenous genes within a cell. A method comprising: a) the gene safe harbor site of a transcription regulatory protein coding gene. The process of targeted insertion into the first allele; and b) Targeted insertion of the induction cassette into a second allele at the same gene-safe harbor site. A process in which the induction cassette is operably connected to an induction promoter, non-cor The DNA comprises a ding RNA sequence, and the promoter is configured to control the transcriptional regulatory protein. Thus, the non-coding RNA sequence is regulated, and the transcription or translation of endogenous genes is suppressed. The above process, The present invention provides the method including the above.

[0037] Furthermore, the present invention relates to a method for knocking out endogenous genes in cells, a) Genes encoding transcriptional regulatory protein codes and genes encoding Cas9 or its derivatives The process of targeted insertion of the gene-safe harbor site into the first allele; and b) Targeted insertion of the induction cassette into a second allele at the same gene-safe harbor site. A process in which the induction cassette is operably connected to an induction promoter, guide RN A is included, the promoter is regulated by a transcription regulatory protein, and the gRNA sequence is internal The above step involves targeting the causative gene. The present invention provides the method including the above.

[0038] Such a single-step knockout or knockdown is novel and part of the present invention. It can be formed.

[0039] In one embodiment, the present invention relates to a method for forward programming of pluripotent stem cells. , a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A step of targeted insertion of an induction cassette into a second gene-safe harbor site, the same as above The induction cassette operably linked to the induction promoter is a transcription factor encoding a key lineage. The gene sequence is included, and the inducing promoter is regulated by the transcription regulatory protein. The aforementioned process, Includes, The present invention provides the method wherein the first and second gene-safe harbor sites are different.

[0040] Further or additional induction cassettes may be further GSHs separate from the first and second GSHs. It can be inserted into.

[0041] Forward programming of pluripotent stem cells to specific mature cell types is highly desirable. This can be achieved using the dual-targeting platform of the present invention. The specific method for that cell type is described below.

[0042] In one embodiment, the present invention is a method for generating muscle cells from pluripotent stem cells, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A second gene-safe harbor of the MYOD1 gene operably linked to the inductive promoter - A step of targeted insertion into a site, wherein the inducing promoter is directed to the transcription regulatory protein The above process is thus adjusted, It includes, and the first and second gene-safe harbor sites are different, The present invention provides a method comprising culturing the cells in the presence of retinoic acid.

[0043] The MYOD1 gene is a myogenesis differentiation 1 protein-coding gene. Preferably, retinoin The acid (RA) is all-trans RA.

[0044] In another embodiment, the present invention relates to a method for generating muscle cells from pluripotent stem cells expressing MYOD1. The present invention provides a method comprising culturing the cells in the presence of retinoic acid. .

[0045] Preferably, the RA is all-trans RA. Preferably, the cells overexpress MYOD1. They are doing it.

[0046] In a further embodiment, the present invention relates to a method for generating oligodendrocytes from pluripotent stem cells. It is a law, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) SOX 10, OLIG2, NKX2.2, and NKX6.2 genes operably linked to inductive promoters A step of targeted insertion into a second gene-safe harbor site of any combination thereof, The inductive promoter is regulated by the transcription regulatory protein, in the above step, The present invention provides the method comprising the first and second gene-safe harbor sites, wherein the first and second gene-safe harbor sites are different.

[0047] The SOX 10, OLIG2, NKX2.2, and NKX6.2 genes are related to the transcription factors SOX 10, OLIG2, and NKX2, respectively. It codes for .2 and NKX6.2. [Brief explanation of the drawing]

[0048] [Figure 1](a-d): Validation of an optimized dual genome safe harbor targeted overexpression system. Figure 1(a) Design of gene targeting vectors for hROSA26 and AAVS1 loci. HAR: homology arm, SA: splice acceptor, T2A: T2A ribosome skipping signal; Neo: neomycin resistance gene; Puro: puromycin resistance gene; pA: polyadenylation signal; CAG: constitutively active CAG promoter; rtTA: third-generation rtTA; TRE: inducible Tet response element; EGFP: highly sensitive green fluorescent protein. Figure 1(b) shows EGFP induction and rescue dynamics in EGFP-expressing hESCs detected by flow cytometry (median fluorescence intensity, MFI) (Figure 1(c)). Results are from two biological copies at each time point and are expressed as mean ± SEM. All values ​​were normalized to the maximum fluorescence intensity 5 days after doxycycline (referred to as day 0 in the figure). Figure 1(d) shows the dose-response of doxycycline to EGFP overexpression in EGFP-expressing hESCs after 5 days of doxycycline induction. Results are from two biological replicas for each condition and are expressed as mean ± SEM. All values ​​were normalized to the maximum fluorescence intensity measured experimentally. EGFP expression levels in GSH-targeted constitutive CAG-EGFP hPSCs and EGFP expression levels in dual GSH-targeted inducible TRE-EGFP hPSCs after doxycycline induction. Wild-type hPSCs and non-inducible TRE-EGFP cells were included as negative controls. [Figure 2](a-d): Overview of the experimental approach and results of the rapid single-step conversion of hPSCs to neurons (i-neurons) after doxycycline (dox) treatment. Figure 2(a) is a schematic diagram of this conversion, in which cells transformed with NGN2 according to the present invention are induced to differentiate into neurons after Dox treatment. Figure 2(b) shows the forward programming time course of i-neuron generation from hESCs as shown by quantitative RT-PCR analysis, and shows the temporal expression patterns of panneuron marker genes (MAP2, SYP), forebrain marker genes (BRN2, FOXG1), and glutamate neuron marker genes (VGLUT2, GRIA4). Cells were analyzed on the indicated day of doxycycline treatment. Values ​​are shown compared to the endogenous housekeeping gene PBGD and are normalized for pluripotency. Results are from three biological replicas at each time point and are expressed as mean ± SEM. Figure 2(c) shows the quantification of βIII tubulin (TUBB3)-positive neurons by immunostaining in i-neurons derived from hESCs one week after induction. Using undifferentiated cells as a negative control, we report the figures for i-neuron generation after 25 passages (+P25) in newly isolated NGN2-expressing hESCs. Figure 2(d) is a series of phase-contrast images showing morphological changes in cells, illustrating the forward programming time course of i-neuron generation from hESCs. [Figure 3](a-d): Forward programming of hPSCs into skeletal muscle cells. Figure 3a shows a schematic diagram of rapid single-step conversion of hPSCs into skeletal muscle cells by inducible overexpression of MYOD1 and treatment with retinoic acid. Figure 3b shows quantitative RT-PCR analysis of the temporal expression patterns of myocyte marker genes during i-myocyte generation from hESCs. All values ​​are shown in comparison to hPSCs. Results are from three biological replicas at each time point and are expressed as mean ± SEM. Figures 3(c) and (d) show quantification of MHC-positive cells by flow cytometry at 10 days post-induction, demonstrating that OPTi-MYOD1 hPSCs retain their myogenic potency even after extended culture periods and after passage (p) following targeted integration of the MYOD1 system. Figures are reported for i-myocyte generation in newly isolated OPTi-MYOD1 hESCs or in the same cells after 50 passages (+P50), using undifferentiated cells as a negative control. [Figure 4](a~f): Targeting strategies for a dual GSH-targeted Tet-ON overexpression system. Figure 4(a) shows an experimental workflow for sequential targeting of the hROSA26 and AAVS1 loci in hPSCs. Keywords: Cas9n: D10A nickase mutant Cas9 endonuclease from S. pyogenes; ZFN: zinc finger nuclease; Neo: neomycin; Puro: puromycin; rtTA: third-generation reverse tetracycline transactivator. This shows an inducible EGFP expression system (i-EGFP). Figure 4(b) shows a schematic diagram of the hROSA26 targeting strategy. Figure 4(c) shows the AAVS1 targeting strategy. Keywords in Figures 4(b) and (c): R26-prom: ROSA26 locus promoter (THUMPD3-AS1 gene); AAV-prom: AAVS1 locus promoter (PPP1R12C gene); ZFN: zinc finger nuclease; 5'-HAR / 3'-HAR: upstream / downstream homology arms; SA: splice acceptor; T2A: T2A peptide; pA: polyadenylation signal; CAG: CMV early enhancer, chicken β-actin and rabbit β-globin hybrid promoter; TRE: Tet response element; EGFP: highly sensitive green fluorescent protein. Figure 4(d) shows a schematic diagram of the genotyping strategy used to identify correctly targeted hROSA26 and AAVS1 targeted hPSC strains; GSH-prom: GSH promoter (hROSA26 and AAVS1, respectively); WT: wild type; induction cassette: all exogenous sequence incorporated after targeting. Locus PCR: PCR spanning a target locus using two primers that exclusively bind to the genomic DNA outside the genome sequence corresponding to the homology arm. Note that due to its high GC content, the CAG promoter cannot be amplified by normal PCR. Therefore, if the CAG-containing expression cassette is correctly inserted, the PCR amplicon will disappear. The presence of a wild-type band indicates the presence of a non-targeting allele; the disappearance of the wild-type band indicates homozygous targeting. 5'-INT / 3'-INT PCR: PCR spanning the 5' and 3' insertion sites, respectively. A PCR amplicon of the correct size indicates correct integration.3'BB PCR: PCR spanning homology arms / targeting vector skeleton junctions. The presence of PCR products indicates untargeted, nonspecific integration of the donor plasmid. Figure 4(e) is a photograph of the gel showing the genotyping results for selected hROSA26-CAG-rtTA targeted heterozygous (HET) and homozygous (HOM) H9 hESCs. Figure 4(f) is a photograph of the gel showing the genotyping results for selected AAVS1-TRE-EGFP targeted heterozygous (HET) and homozygous (HOM) H9 hESCs. 1kb+: 1kb plus DNA ladder; WT: wild-type hESC; PL: targeted plasmid; H2O: water control. [Figure 5](a~e): Development of an optimized inducible overexpression platform (OPTi-OX) based on hPSC dual GSH targeting. Figure 5(a) shows that dual GSH targeting-inducible EGFP H9 hESCs were pooled into four experimental groups depending on whether one or both alleles of the hROSA26 and AAVS1 loci were successfully targeted. Figure 5(b) shows the detection of rtTA protein by Western blotting in successfully targeted heterozygous and homozygous H9 hROSA26-CAG-rtTA hESCs. Human ESCs carrying second-generation rtTA at random genomic locations were included as control samples. α-tubulin: loading control. Figure 5(c) shows flow cytometry analysis of representative examples of the various dual GSH targeting-inducible EGFP hESCs described in Figure 5(a). Figure 5(d) shows the median fluorescence intensity (MFI) of EGFP expression in the various dual GSH targeting-inducible EGFP hESCs described in Figure 5(a). Cells were analyzed by flow cytometry under control conditions (doxycycline-free, CTR) or 5 days after doxycycline treatment (DOX). Each data point represents an individual clonal strain. CAG-EGFP hESCs and wild-type (WT) hESCs were included for comparison. Statistical analysis of the doxycycline-treated group (n=4–5, as shown) showed that EGFP expression levels were highest in double homozygous clones (one-way ANOVA with post-Hock-Dunnett test; F(2, 10)=25.34, p=0.0001; **** p<0.0001; ** p=0.0026). This condition was selected for further experiments. Figure 5(e) shows the percentage of EGFP+ cells in various double GSH-targeted i-EGFP hESCs described in Figure 5(a). [Figure 6](a-d): Characterization of the OPTi-OX platform in hPSCs and during germ layer differentiation. Figure 6(a) shows flow cytometry analysis of EGFP levels in normally targeted live hPSCs and after their differentiation into the three germ layers following 5 days of doxycycline treatment. The acquisition setting was configured to include induced high levels of EGFP expression (DOX). The non-inducible control population (CTR) is located immediately to the left of the y-axis. Figures 6(b) and 6(c) show an overview of the flow cytometry plots of 6(a), including median fluorescence intensity (MFI) and the percentage of EGFP+ cells. Figure 6(d) shows bar graphs of quantitative RT-PCR results for EGFP mRNA expression levels in homozygous pluripotent stem cells and EGFP mRNA expression levels after differentiation into the three germ layers. WT: wild type. [Figure 7] Characterization of human i-neurons. Quantitative RT-PCR results show rapid downregulation of pluripotency factors NANOG and OCT4 upon doxycycline treatment. [Figure 8] RA signaling during myocyte induction. This figure shows qPCR analysis of six retinoids and retinoid receptors during myocyte induction, demonstrating that RARα, RARβ, and all three RXR isoforms are expressed throughout the i-myocyte induction process, but RARγ is not. A is α, B is β, and G is γ. [Figure 9]Figures 9(a)-9(c): Characterization of OPTi-MYOD1 hESC development into human i-myocytes. Figure 9(a) shows the time course of forward programming of OPTI-MYOD1 hPSCs into induced myocytes. Morphological changes were demonstrated by automated phase-contrast images acquired every 30 minutes using the Nikon Biostation IM time-lapse system. Scale bar: 200 μm. Figure 9(b) shows the results of qPCR (left graph) demonstrating rapid downregulation of pluripotency factors NANOG and OCT4 upon doxycycline treatment of OPTi-NGN2 hESCs. All five major human skeletal muscle cell-specific myocyte heavy chain isoforms (encoded by the MYH gene family) are strongly upregulated during myocyte forward programming (right graph). These include two isoforms expressed during embryonic development and postnatal muscle development (embryonic isoform MYH3; neonatal isoform MYH8), and three isoforms normally expressed in adult skeletal muscle [MYH7 in slow-twitch (type I) fibers; MYH2 in fast-twitch fatigue-resistant (type IIa) fibers; and MYH1 in fast-twitch, fatigue-resistant (type IIx) fibers]. In contrast, MYH4, which represents the MHC isoform that makes up fast-twitch, fast-twitch, fatigue-resistant muscle cell fibers in cats, is not expressed in significant amounts (less than 1%) in humans and is not induced during the forward programming time course. Figure 9(c) shows induced skeletal muscle cells that express a wide range of typical marker proteins, including F-actin (visualized via AlexaFluor488-conjugated phalloidin toxin), neuronal adhesion molecules (NCAM), desmin (DES), myosin heavy chain (MYH), titin (TTN), α-actinin (ACTN2), and troponin T (TNNT), but do not express the myoblast progenitor cell markers PAX3 and PAX7. All samples were counterstained with myogenin (MYOG). Scale bar: 50 μm. DAPI: nuclear staining. [Figure 10]These three graphs show the results of qPCR for total MYOD1, endogenous MYOD1, and MYOG at 2 days after induction of OPTi-MYOD1 hPSCs at different doxycycline concentrations. The results of qPCR at 48 hours after induction at different doxycycline concentrations are also shown. Expression is plotted against the endogenous housekeeping gene PBGD. [Figure 11] Diagram of the Tet-ON system. Tet-ON consists of two components: at the top, an activator cassette is shown in which a constitutive promoter (cP) promotes the expression of rtTA (reverse tetracycline trans-activator). rtTA is a fusion protein consisting of a mutant of the prokaryotic Tet repressor (TetR) and the transcriptional trans-activator domain VP16 (derived from herpes simplex virus). At the bottom, the response domain is shown. This consists of an inductive promoter (TRE, Tet response element) and the gene of interest. The TRE is an artificial promoter that responds to rtTA. It consists of seven consecutive tet operons (tetO7) and a strong minimal CMV promoter (mCMV), which is inactive on its own and recruits the transcriptional mechanism only when rtTA is bound to the seven tet operons. Doxycycline, a tetracycline derivative, is required for the binding of the mutant TetR to the TRE, resulting in the expression of the inductive cassette, in this case EGFP (pA: polyadenylation signal). [Figure 12](a-d) Forward programming of hPSCs to oligodendrocytes. Figure 12(a) shows a schematic diagram of the experimental approach to the rapid conversion of OPTi-OLIG2-SOX10 hPSCs to oligodendrocyte lineage cells (i-OPC and i-OL). Figure 12(b) shows the quantification of BrdU-positive cells after three consecutive passages every four days and the accompanying BrdU pulses lasting for four days each (P = passage number). Figure 12(c) shows quantitative RT-PCR analysis of the time-course expression patterns of myelin-related protein (CNP, MAG, MBP, MOG, and PLP) coding genes during i-oligodendrocyte generation from hPSCs. OPTi-OLIG2-SOX10 hPSCs were induced in oligodendrocyte medium supplemented with PDGF-aa and FGF2. Mitogens were removed one week after induction to enable terminal differentiation. All values ​​are shown normalized to pluripotency relative to the endogenous housekeeping gene PBGD. Results are from 2-3 biological replicas at each time point and are expressed as mean ± SEM. Figure 12(d) shows the quantification of CNP and PLP-positive cells by immunostaining in i-oligodendrocytes derived from OPTi-OLIG2-SOX10 hPSCs 20 days after induction. Using undifferentiated cells as a negative control, we report figures for i-oligodendrocytes after 50 passages (+P50) in newly isolated OPTi-NGN2 hPSCs. [Figure 13] This is a schematic diagram of the principle of the present invention. Essentially, it illustrates insertions into two different gene-safe harbor sites in the core of the present invention. One insertion controls the expression of a gene sequence within the induction cassette in the second insertion. As shown, additional genetic material can be included in the polycistronic vector construct. Furthermore, three or more gene-safe harbor sites can be targeted so that multiple induction cassettes or other genetic material can be placed under the control of a regulator located at the first GSH site. [Figure 14]Figures (a-f) show the results illustrating the progress of the inducible knockdown system based on dual GSH targeting of hSPCs. Figure 14a shows the experimental approach: H1- H1 promoter, TO- tet operon, tetR- tetracycline repressor. Figure 14b is a schematic diagram of the transgenic alleles created to obtain hESCs expressing an EFGP reporter transgene that can be silenced using inducible EGFP shRNA. Figure 14c shows EGFP expression in hESCs targeted with the indicated combinations of inducible EGFP shRNA and tetR for 5 days in the absence or presence of tetracycline (STD = wild-type standard, OPT = codon optimized). Dual-targeted hESCs lacking EGFP shRNA were used as negative controls. For the same tetR strain without tet or shRNA, ns=p>0.05 (not significant), **=p>0.01, ***=p>0.001. Figure 14d shows representative Western blots of tetR in ROSA26-targeted hESCs expressing STD or OPT tetR. HET = heterozygous targeting, HOM = homozygous targeting. hESCs with random incorporation of STD tetR are shown as a positive criterion, while WT h9 hESCs are a negative control. TUB4A4A is a loading control. Various protein amounts were added to facilitate quantitative comparison. Figure 14(E): EGFP knockdown and rescue kinetics in EGFP OPTiKD hESCs as measured by flow cytometry (MFI) and qPCR (mRNA). Results are from two independent cultures at each time point. Figure 14(F): Tetracycline dose-response curves of EGFP knockdown in EGFP OPTiKD hESCs. Semi-maximal inhibitory concentration (IC50) is reported. Results are from two independent cultures per dose and show mean values. [Figure 15](a, b, and c) Verification of ROSA26 and AAVS1 loci as true GSH. Figure 15a shows an experimental approach for the construction of GSH EGFP reporter hPSCs to test GSH expression during differentiation. Neurons, oligodendrocytes, and astrocytes were obtained in bulk cultures containing mixtures of these cell lines, and all other cell types were constructed individually. Figure 15b is a schematic diagram of the ROSA26 and AAVS1 EGFP reporter transgenic alleles. R26-prom: ROSA26 locus promoter; AAV-prom: AAVS1 locus promoter; 5'-HAR / 3'-HAR: upstream / downstream homology arms; SA: splice acceptor; T2A: autocleaved T2A peptide; Neo: neomycin resistance; Puro: puromycin resistance; pA: polyadenylation signal; CAG: CAG promoter; EGFP: highly sensitive green fluorescent protein. Figure 15(C): EGFP expression in hESCs targeted with the indicated combinations of inducible EGFP shRNA and tetR (wild-type standard tetR, STDtetR, or codon-optimized tetR, OPTtetR) over 5 days in the absence or presence of tetracycline. Dual-targeted hESCs lacking EGFP shRNA were used as negative controls. Results are from 2-3 individual strains per condition (Table 1). For the same tetR strains without tet or shRNA, ns=p>0.05 (not significant), **=p<0.01, ***=p<0.001 (ANOVA with post-hoc Holm-Sidak comparison). [Figure 16](a~d) Construction of ROSA26 and AAVS1 EGFP reporter hESCs. Figure 16(A): Schematic diagram of the ROSA26 targeting approach and the genotyping strategy used to accurately identify target strains. Cas9n: D10A nicasse mutant Cas9 endonuclease from S. pyogenes. R26-prom: ROSA26 locus promoter (THUMPD3-AS1 gene); 5'-HAR / 3'-HAR: upstream / downstream homology arms; transgene: region incorporated after gene targeting; locus PCR: PCR product of the wild-type ROSA26 locus (shows untargeted allele); locus PCR / allele loss: PCR product of the targeted allele / PCR that fails when the transgene contains a GC-rich CAG promoter (shows expected transgene targeting); 5'INT / 3'INT PCR: PCR product of the 5' / 3' end of the transgene integration region (shows expected transgene targeting); 5'BB / 3'BB PCR: PCR product of the 5' / 3' end of the vector backbone (shows untargeted, nonspecific plasmid integration). Note that similar targeting and genotyping strategies were applied to the AAVS1 gene coordinate system. Figure 16(B): Schematic diagram of ROSA26 transgenic alleles constructed to test the best strategy for constitutive EGFP (highly sensitive green fluorescent protein) expression. ENDO-EGFP: EGFP promoted by the endogenous ROSA26 promoter (R26-prom; targeted vector pR26-Puro_ENDO-EGFP); EF1α-EGFP: EGFP promoted by the elongation factor 1α promoter (targeted vector pR26-Neo_EF1α-EGFP); CAG-EGFP: EGFP promoted by the CAG promoter (targeted vector pR26-Neo_CAG-EGFP); SA: Splice acceptor; Puro: Puromycin resistance (puromycin N-acetyltransferase); Neo: Neomycin resistance (neomycin phosphotransferase II); pA: Polyadenylation signal.Figure 16(C): Percentage of EGFP-positive cells (EGFP+; gated) and median EGFP fluorescence intensity (MFI) or flow cytometry quantification of wild-type H9 hESCs in representative ROSA26-EGFP reporter hESC clones. Figure 16(D): Percentage of EGFP-positive cells in ROSA26-EGFP reporter hESCs. Results are for three clones with heterozygous ROSA26 targeting for each condition. [Figure 17] Validation of an optimized inducible knockdown platform after hPSC differentiation. The plots show EGFP expression, measured by qPCR in the indicated cell types derived from EGFP OPTiKD (iKD) and sOPTiKD (siKD) hESCs, under tetracycline absence (CTR) or presence (TET) for 5 days. EGFP levels are reported for each individual strain against control conditions in the same strain. Abbreviations indicate the strains shown in Figure 15 (pluri: undifferentiated). Results are from two independent cultures for each condition. [Figure 18] (a-d) Development of an inducible CRISPR / Cas9 knockout platform optimized for hPSCs. Figure 18a shows an experimental approach for the creation of inducible knockout (iKO) hPSCs. Figure 18b shows a schematic diagram of the cloning procedure for creating an AAVS1 targeting vector with an inducible gRNA cassette. Figure 18c shows transgenic alleles created to obtain hESCs expressing an EGFPd2 reporter transgene that can be knocked out by CRISPR / Cas9 using inducible EGFP gRNA (EGFP sOPTiKO hESC). Bsd: blastosidine resistant; EGFPd2: unstable EGFP. Figure 18(d): Flow cytometry quantification of EGFPd2 inducible knockout dynamics in sOPTiKO cells from Figure 19c (gRNA 2-TO) and b (gRNA 3-2TO). The percentage of EGFP-positive cells was monitored daily after tetracycline addition. Results are from two independent cultures. [Figure 19](a~e): Development of an optimized inducible CRISPR / Cas9 knockout platform in hESCs. (A~D) show representative flow cytometry of EGFPd2 expression in EGFPd2 homozygous sOPTiKO hESCs possessing the indicated combinations of gRNA (2 or 3) and inducible promoter (TO or 2TO, see Figure 19e). Targeting vectors: pAAV-Puro_siKOEGFP-2(19a), pAAV-Puro_siKO-2TO-EGFP-2(19b), pAAV-Puro_siKO-EGFP-3(19c), pAAV-Puro_siKO-2TO-EGFP-3(19d). Cells were cultured for 5 days in the presence of tetracycline (TET) or maintained under control (CTR) conditions in the absence of tetracycline. Note that the histograms were normalized so that the area under the curve equals 1 (100%) for all presented samples to facilitate direct visual comparison. Figure 19(e): Nucleotide sequences of inducible H1 Pol III promoters for sOPTiKO systems containing one or two tet operons (H1-TO and H1-2TO, respectively). Key sequence features are highlighted. Restriction enzyme cleavage sites used for gRNA cloning are shown (Figure 18B). DSE: distal sequence element. PSE: proximal sequence element; TETO2: tet operon; +1: start site of RNA transcription. [Figure 20] This is a depiction of the plasmid map used in the examples of this application: pSpCas9n(BB)_R26-R. [Figure 21] This is a depiction of the plasmid map used in the examples of this application: pSpCas9n(BB)_R26-L. [Figure 22] This is a depiction of the plasmid map used in the examples of this application: pR26_CAG_EGFP. [Figure 23] This is a depiction of the plasmid map used in the examples of this application: pR26_CAG_rtTA. [Figure 24] This is a depiction of the plasmid map used in the examples of this application. pZFN-AAVS1-L-ELD (zinc finger nuclease, left). [Figure 25] This is a depiction of the plasmid map used in the examples of this application. pZFN-AAVS1-R-KKR (zinc finger nuclease, right). [Figure 26] This is a depiction of the plasmid map used in the examples of this application: pAAV_CAG_EGFP (donor). [Figure 27] This is a depiction of the plasmid map used in the examples of this application: pR26-Neo_CAG-OPTtetR (hROSA26-targeted codon-optimized tetR). [Figure 28] This is a depiction of the plasmid map used in the examples of this application: pAAV-Puro_iKD (AAVS1 targeting of inducible shRNA). [Figure 29] This is a depiction of the plasmid map used in the examples of this application: pAAV-Neo_CAG-Cas9 (AAVS1-targeted Cas9). [Figure 30] This is a depiction of the plasmid map used in the examples of this application: pAAV-Puro_siKO (AAVS1 targeting of inducible gRNA). [Figure 31] This is a depiction of the plasmid map used in the examples of this application: pAAV-Puro_siKO-2TO (AAVS1 targeting of inducible gRNA, version with two tet operons in the promoter). [Figure 32] This is a depiction of the plasmid map used in the examples of this application: pAAV_TRE-EGFP (EGFP-inducible overexpression, attached). [Figure 33] This is a depiction of the plasmid map used in the examples of this application: pAAV_TRE-MYOD1 (MYOD1-inducible overexpression for muscle). [Modes for carrying out the invention]

[0049] (Detailed explanation) The present inventors have found that in eukaryotic cells, particularly pluripotent stem cells and their offspring, within the induction cassette... We developed a method useful for inducing the transcription of the included gene sequences.

[0050] This method involves the overexpression of induction cassettes within stem cells that promote the development of specific mature cell types. Through this process, it is particularly applicable to forward programming of pluripotent stem cells. Furthermore, the function To study loss or to alter the function or behavior of cells in these cells, It also applies to the knockdown or knockout of endogenous functions within cells. Knockout refers to the DNA sequence encoding a protein-coding gene or non-coding RNA. It can be applied to the following: In all cases, by knockout or knockdown, the method of the present invention They can be targeted.

[0051] This method uses a system for inducing transcription that can be divided into two or more GSHs, and is used to induce the growth of stem cells. This is based on at least dual targeting of safe harbor areas within the NM. However, This method is not limited to stem cells, but can be used, for example, in research or gene therapy. It can be used to modify the genome of the cell type. In the method of the present invention, one The GSH contains genes contained within an induction cassette that is inserted into different GSHs at other locations in the genome. It is modified to contain transcription factors necessary to induce sequence transcription. The offspring is preferably constitutively expressed. Exogenous substances / drugs affect the activity of transcription regulatory proteins. It must be controlled, and therefore supplied to control the expression of the induction cassette. This is preferable. Since at least two separate GSHs are used in the method of the present invention, each GSH is doubled. Since it exists on both chromosomes of a living organism, there are a total of four possible insertion loci. When all four gene loci are modified using the method of the present invention, the potential for the cells to... This increases the amount of transcription. An example of various results of targeted insertion is shown in Figure 5a. Furthermore, the present invention This method uses at least two different GSH sites. Further GSH sites may lead to further transformation. Regulatory factors, induction cassettes or select markers, antibiotics or drug resistance genes, CRISPR This includes, but is not limited to, genes related to the Cas9 system or genes with unknown functions. It is understood that it can be used to introduce any other genetic material.

[0052] Therefore, the present invention relates to a method for controlling the expression of an inserted gene sequence within a cell, a) Targeted insertion of transcriptional regulatory protein-coding gene sequences into the first gene-safe harbor site. The process of entry; and b) A step of targeted insertion of an induction cassette into a second gene-safe harbor site, the same as above The induction cassette includes the gene sequence operably linked to the induction promoter, The promoter is regulated by the transcription regulatory protein, in the above step, Includes, The present invention relates to the method wherein the first and second gene-safe harbor sites are different.

[0053] Furthermore, in this embodiment of the present invention, further identical or different induction cassettes are the first and It can be inserted into a second GSH and a further GSH different from the second GSH. Such induction cassettes are described herein. As described in [the document].

[0054] Specific insertion into gene-safe harbor regions is considered a safer form of genome modification. This is expected to be preferable to random genome integration, and is a natural gene expression symmetric It can cause undesirable side effects, such as mutations that lead to reniculation or cancer cell types. The possibility is low.

[0055] Gene safe harbor (GSH) sites are areas where a gene or other genetic material is inserted into a cell or is inserted into a cell. An insertion locus is a gene locus within the genome that can be inserted without causing any adverse effects on the genetic material. The expression of the gene sequence is not disrupted by arbitrary read-through expression from adjacent genes, and induction cassette The most beneficial GSH site is one where the expression of the gene minimizes interference with the endogenous transcription program. A more formal approach to help determine whether a specific gene locus will be a GSH site in the future. Criteria have been proposed (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73-8). (doi:10.1038 / nbt.1717.) These criteria include (i) at least 50kb from the 5' end of any gene. (ii) a site that is 300kb or more from any gene related to cancer, (iii) any (iv) A region of microRNA (miRNA) that is 300kb or longer, located outside the transcription unit. This includes (v) regions located outside the superconserved region (UCR). Since GSH does not meet all the criteria, it is not possible to meet all of these proposed criteria. It may not be necessary. A suitable GSH will meet at least two, three, four, or all of these criteria. It is thought that...

[0056] Further sites are those that the virus naturally incorporates without interrupting natural gene expression. It can be identified by searching.

[0057] Any suitable GSH site allows for the insertion of genetic material without harmful effects on the cell. The method of the present invention is intended to enable the transcription of genetic material. Yes, it is possible. A person skilled in the art can use this simplified criterion to determine the appropriate GSH, and / or more than the above. Formal standards can be identified.

[0058] Regarding the human genome, several GSH sites have been identified, and these are associated with the AAVS1 gene. This includes the gestational locus, the hROSA26 locus, and the CLYBL gene. The CCR5 gene and HPRT gene are also included. Discussed as a possible GSH, further investigation suggests that one or more of these are present in the human genome. It can be identified as a GSH.

[0059] The adeno-associated virus integration site 1 (AAVS1) is a protein on human chromosome 19. It is located within the gene for phosphate phosphatase 1, regulatory subunit 12C (PPP1R12C), and is distributed evenly in human tissues. It is expressed unilaterally and ubiquitously. This site functions as an integration locus specific to AAV serotype 2. Therefore, it was identified as a possible GSH. AAVS1 has an open chromatin structure and Natural chromosome insulators that enable resistance to silencing of induction cassettes Because it contains [the specified element], it has been shown to be a good environment for transcription. Disruption of the PPP1R12C gene. There are no known side effects on cells resulting from this. Furthermore, the induction cassette inserted into this site The ret remains transcriptionally active in many diverse cell types. Therefore, AAVS 1 is considered to be GSH and has been widely used in targeted genetic recombination in the human genome. Ta.

[0060] The hROSA26 site is related to the mouse GSH (ROSA26-reverse splice acceptor site #26). It was identified based on sequence similarity. An orthologous region was identified in humans, but this region is... It is not commonly used for induction cassette insertion. The inventors have identified a target specific to the hROSA26 site. We developed a synthesis system, which allowed us to insert genetic material into this gene locus. hROS The A26 locus is located on chromosome 3 (3p25.3) and can be found in the Ensembl database. This is possible (GenBank:CR624523). The exact genome coordinates of the integration site are 3:9396280-93963 03: Ensembl. The integration site is the openly linked long non-coding RNA of THUMPD3. It is located within the ORF (inverse chain). The hROSA26 site has an endogenous promoter. Therefore, the inserted genetic material can utilize its endogenous promoter, or the promo It can be inserted so as to be operably connected to the motor.

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

[0062] CCR5, located on chromosome 3 (position 3p21,31), encodes the major coreceptor for HIV-1. It is a gene. The idea of ​​using this region as GSH is not thought to have any adverse effects. However, this arises from a null mutation in this gene that predisposes the individual to HIV-1 infection resistance. Because zinc finger nucleases targeting this gene locus have been developed, The insertion of genetic material has become possible. The natural function of CCR5 still needs to be elucidated. Considering this, this area remains a potential GSH that could be useful for the present invention. .

[0063] The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene is pre Tranexa plays a central role in the production of purine nucleotides via the salvage pathway. It encodes the enzyme spherase. Therefore, insertion at this site disrupts normal cellular function. Further work is needed to ensure that it does not break. However, The GSH site is debatable. Insertion at this site is a modification for gene therapy. This could be more applicable to any mature cell type.

[0064] GSH has been identified in other organisms, and in mice, it is found at the ROSA26, HRPT, and Hipp11 (H11) loci. The mammalian genome may contain GSH sites based on pseudo-attP sites. Regarding this, it is possible to incorporate an induction cassette-containing plasmid possessing the attB site into the pseudo-attP site. Because it possesses power, hiC31 integr, a recombinase derived from streptomyces phage, is effective. Lase was developed as a non-viral insertion tool.

[0065] GSH is also present in the plant genome, and the modification of plant cells constitutes part of this invention. Yes, it is possible. GSH has been identified in the rice genome (Cantos et al., Front. Plant). Sci., 26 June 2014, Volume 5, Article 302, http: / / dx.doi.org / 10.3389 / fpls.2014.0 0302 ).

[0066] In the method of the present invention, since insertion occurs in different GSHs, at least two GSHs are present in the present invention. This method requires the first GSH to be modified by inserting a transcription regulatory protein. GSH is an insertion of an induction cassette containing a gene sequence operably linked to an induction promoter. Modified by insertion. Other genetic material may also be inserted along with one or both of these elements. It can be introduced. The gene sequence operably linked to the induction promoter in the induction cassette is Preferably, it is a DNA sequence. The gene sequence(s) of the induction cassette encode an RNA molecule. Therefore, it is transcribable. Transcription is controlled using an inducible promoter. RNA molecules are, It can be any sequence, but preferably a protein-coding mRNA, shRNA, or g It is RNA.

[0067] The first GSH can be any suitable GSH site. Optionally, it is constitutively expressed within This is a GSH with a generative promoter. This is constitutive to the inserted transcription regulatory protein. It brings about expression. The suitable GSH is the hROSA26 site for human cells. Alternatively, the insertion site The regulatory protein is operably linked to a promoter, preferably a constitutive promoter. The constitutive promoter can be used in combination with insertion at the hROSA26 site. Cut.

[0068] Transcription regulatory proteins are DNA, preferably a DNA portion located in or near the promoter. It is a protein that binds specifically to the position of the sequence, and is involved in the binding of the promoter of the transcription machinery, and consequently This involves either promoting the transcription of the DNA sequence (as a transcription activator) or inhibiting this process. (Transcriptional repressors). Such entities are also known as transcription factors.

[0069] The DNA sequence to which a transcription regulatory protein binds is called a transcription factor binding site or response element. These are found within or near the promoter of regulatory DNA sequences.

[0070] Transcriptional activating proteins bind to response elements and promote gene expression. Such proteins are preferred in the present invention's method for controlling the expression of induction cassettes.

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

[0072] Transcriptional regulatory proteins are involved in the binding of substances and interactions with other transcription factors (e.g., homozygous or... (This includes heterodimerization) or co-regulatory proteins, phosphorylation, and / or methylation. Transcription regulators can be activated or inactivated by that mechanism. Therefore, it can be controlled.

[0073] If a transcription regulatory protein is a transcription activating protein, then the transcription activating protein is active. It is preferable that this requires modification. This activation can be carried out by any suitable means, but transcription The nodal protein is preferably activated by the addition of exogenous substances to the cell. The supply of exogenous substances to this can be controlled, thus inhibiting the activation of transcription regulatory proteins. It can be controlled. Alternatively, exogenous substances can inactivate transcription regulatory proteins. It can be supplied and then removed to activate transcription regulatory proteins. Cut.

[0074] If a transcription regulatory protein is a transcription repressor protein, the transcription repressor protein is inactivated. It is preferable that this is required. Therefore, the substance is such that the transcription repressor protein represses transcription. It is supplied to prevent this from happening, and therefore, transcription becomes possible.

[0075] Any suitable transcription regulatory protein is preferably one that can be activated or deactivated. Exogenous substances may be used. Exogenous substances may be supplied to control transcription regulatory proteins. Preferred. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins. .

[0076] Tetracycline-controlled transcriptional activation is caused by the antibiotic tetracycline or its induction. In the presence of one type of body (e.g., the more stable doxycycline), transcription is reversibly initiated. This is a method of inducible gene expression that either activates or inhibits it. In this system, transcription-activating proteins The substance is a tetracycline-responsive transcription-activating protein (rtTa) or a derivative thereof. The rtTA protein can bind to DNA via a specific TetO operator sequence. Some repeats of a TetO sequence, such as the one described above, are upstream of a minimal promoter (such as the CMV promoter). They are positioned and together form a tetracycline response element (TRE). The addition of or its derivatives activates (Tet-On) or inactivates (Tet-Off) the rtTA protein. Depending on whether or not this applies, there are two forms of this system.

[0077] In the Tet-Off system, tetracycline or its derivatives bind to rtTA, and rtTA This inactivates the TRE sequence, preventing it from binding, thereby controlling the TRE sequence. This prevents the transfer of genes. This system was first described in Bujard et al.'s paper (1992). Proc. Natl. Ac This was described in ad. Sci. USA 89 (12): 5547-51.

[0078] The Tet-On system consists of two components: (1) constitutively expressed tetracycline-responsive transcriptional activity In the sexualized protein (rtTa) and the rtTA-sensitive inducing promoter (Tet response element, TRE) It is composed of such tetracycline or doxycycline (DOX). It binds to a more stable derivative, leading to the activation of rtTa and enabling it to bind to the TRE sequence. This may induce the expression of TRE regulatory genes. This use is preferred in the method of the present invention. This system is illustrated in Figure 11.

[0079] Therefore, transcription regulatory proteins, in this way, are supplied with externally available antibiotics. Tetrasa which can be activated or deactivated by cyclin or one of its derivatives It may be an iclinate-responsive transcription-activating protein (rtTa) protein. Transcriptional regulatory protein. If the quality is rtTA, the inducing promoter inserted into the second GSH site is tetracycline It includes a response element (TRE). The substance supplied from an external source is the antibiotic tetracycline. or one of its derivatives.

[0080] Mutant and modified rtTa proteins can be used in the method of the present invention, and these include Te t-On highly trans-activating factor (also known as rtTA2S-M2) and Tet-On 3G (from rtTA2S-S2) This includes the rtTA-V16 (also known as the previous rtTA-V16).

[0081] Tetracycline response elements (TREs) are generally found together with minimal promoters. It consists of seven repeats of a 19bp bacterial TetO sequence isolated by the ser sequence. Minimal promoter Since it can be any suitable promoter, it is possible to create variants and modify the TRE sequence. Preferably, the minimal promoter has an expression level of 0 or minimum in the absence of rtTa binding. This indicates that the inducing promoter inserted into the second GSH may include a TRE.

[0082] The modified system based on tetracycline control is the T-REx® system (Thermofishe (r Scientific) and the transcription regulatory protein among them is the transcription repressor protein TetR. The components of this system include (i) a potent early stage human cytomegalovirus (CMV) Inductive promoter including a motor and two tetracycline operators 2 (TetO2) It includes the site and the Tet inhibitor (TetR). The TetO2 sequence is divided into two base pairs of spacers. The 19 nucleotide sequence that is separated [ka] It consists of two copies. In the absence of tetracycline, the Tet inhibitor is an inducible promoter. - Forms homodimers that bind with very high affinity to each TetO2 sequence, and promoter - It inhibits transcription from. When added, tetracycline homodimericly inhibits each Tet repressor. It binds to the body with high affinity, preventing it from binding to Tet operators. Tet inhibitor: The tetracycline complex then dissociates from the Tet operator, enabling expression induction. In this case, the transcription regulatory protein is TetR, and the inducing promoter is two TetO2 sites. It includes [the substance]. The externally supplied substance is tetracycline or a derivative thereof.

[0083] The present invention further relates to codon optimization tetR (OPTtetR), which is described herein. It may be used in any manner, or for any further use where induction or promotion is desired. The actual entity was constructed using multi-parameter optimization of the bacterial tetR cDNA sequence. OPTtetR This allows for a 10-fold increase in tetR expression compared to the standard sequence (STDtetR). Homozygous OPTtetR expression of tetR is sufficient to prevent shRNA leakage, and in the example, Maintain knockdown induction. The sequence of OPTtetR is shown in this specification along with the standard sequence for comparison. Include in the book. This sequence must have at least 75%, 80%, 85%, or 90% homology, more specifically... Specifically, sequences having 91, 92, 93, 94, 95, 96, 97, or 99% homology are patented in this specification. The following residues have been shown to be different between STDtetR and OPTtetR, as indicated in the sequence. These residues are considered important for improving the properties, and therefore, the role of OPTtetR It is preferable not to change any derivative. Any derivative may optionally be at the indicated position These modifications are retained in place.

[0084] Other induced expression systems are known and can be used in the method of the present invention. This is Agilent Technologies' Complete Control Inducible System (SQUID). It contains em). This is Drosophila melongaster ecdysone. The gene for the receptor (EcR) and the inducible promoter containing the binding site for the ecdysone receptor Insect hormones can activate transcription in mammalian cells transfected with both. Based on mon's ecdysone or its analog, ponasterone A (ponA). EcR is a nuclear receptor. It is a member of the retinoid X receptor (RXR) family. In humans, EcR is ecdiso It forms a heterodimer with RXR, which is bound to the response element (EcRE), in the absence of PonA. Transcription is repressed by heterodimers.

[0085] Therefore, the transcription regulatory protein is an inhibitory protein such as an ecdysone receptor or its derivative. It can be an protein. An example of the latter is the DNA-binding domain of EcR, the glucocorticoid receptor. Agilent technologies are a fusion of the transcriptional activation domain of herpes simplex virus VP16. The company's VgEcR synthesis receptor is included. The inducing promoter, along with the minimal promoter, contains EcR It includes the E sequence or a modified version thereof. The modified version is an Agi sequence with mutations. It contains an E / GRE recognition sequence from lent technologies. This E / GRE recognition sequence contains a retinoid X receptor The substance (RXR) includes an inverse half-site recognition element and a GR binding domain. All permissions In the procedure, the substance supplied from the outside is an EcR or to the inductive promoter. Ponasterone A is a derivative that removes the inhibitory effect of its derivatives, allowing transcription to occur.

[0086] Alternatively, the induction system may use the synthetic steroid mifepris as an externally supplied substance. It may be based on ton. In this situation, the DNA-binding domain of the yeast GAL4 protein, human progesterone The cleaved ligand-binding domain (LBD) of the sterone receptor and the activation domain of human NF-κB A hybrid transcription regulatory protein based on (AD) is inserted. The regulatory protein is available from Thermofisher Scientific (Gene Switch®). Yes. Mifepristone activates hybrid proteins and the activation sequence (UAS) and Transcription from an inducible promoter containing GAL4 upstream of the adenovirus E1b TATA box is possible. To enable this. This system is described in the literature by Wang, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91. It is described in 8180-8184.

[0087] Therefore, the transcription regulatory protein is either an activating protein or an inhibitory protein. It could be any suitable regulatory protein. A suitable transcriptional activating protein is Tetrasa Icrin-responsive transcription-activating protein (rtTa) or gene-switch hybrid transcription regulation It is a protein. Appropriate inhibitory proteins include rtTA, TetR, or the Tet-Off version of EcR. This includes the transcription regulatory protein, which may be modified or derivatized as needed.

[0088] The inducing promoter contains elements suitable for binding to or interacting with transcription regulatory proteins. It may include. The interaction between the transcription regulatory protein and the inducing promoter is preferably, It is controlled by substances supplied from an external source.

[0089] Any externally supplied substance that binds to or interacts with transcription regulatory proteins is appropriate. It can be a substance. Suitable substances include tetracycline, ponasterone A, and mifepris It includes tons.

[0090] Therefore, the insertion of the first transcription regulatory protein-coding gene into GSH is an induction promoter. - is operably connected and activates an induction cassette inserted into a second different GSH site. To provide a control mechanism.

[0091] Transcriptional regulatory protein genes can be provided for insertion into other genetic material. These substances induce visually identifiable features, including fluorescent proteins and luminescent proteins. This includes genes for marker or reporter molecules such as genes that detect blue light / UV light. Below is the jellyfish green fluorescent protein (GFP) that causes the cells expressing it to emit green light, Lucifer Luciferase, which catalyzes the reaction with phosphorus to produce light, is the red fluorescent protein of the gene dsRed. It contains genes that code for the substance. Such marker or reporter genes are said to The presence of the porter protein supports protein expression from the first GSH, indicating successful insertion. Therefore, it is useful. The selection marker further identifies resistance genes to antibiotics or other drugs. May include: Markers or repositories that enable the study of endogenous (or exogenous) gene expression. It is also possible to introduce a target gene sequence. This makes it possible to excis the desired gene. CasL, Cas9 protein, and, for example, those that act as transcription enhancers or repressors. Cas proteins, including Cas fusion proteins, mediate changes in the expression of other genes. This includes optogenetic tools, tamoxifen-inducible systems such as ERT, and other nuclear tools. Receptor fusion proteins, and designer receptors activated exclusively by designer drugs. Non-inducible expression of molecular tools, including those containing the substance, is sometimes desirable. Furthermore, autoclaving of hormones Mechanisms of cells within the same cell, adjacent cells, or even more distant cells in an organism, including phosphorus or paracrine factors. The sequence encoding the signaling factor that alters function is derived from the same GSH as the transcription regulatory protein. It can be made to manifest.

[0092] Furthermore, additional genetic material may encode non-coding RNA as described herein. It may contain sequences. Examples of such genetic material include miRNAs that can function as gene switches. It contains the gene.

[0093] Transcriptional regulatory protein-coding genes are operablely linked to constitutive promoters. Preferred. Alternatively, the first GSH is the transcription regulatory protein gene and any related genetic material. It is possible to select a constitutive promoter that already has the ability to promote the expression of [the substance]. Constitutive promoters ensure sustained and high levels of gene expression. Human β-acti Promoter (ACTB), cytomegalovirus (CMV), elongation factor 1α (EF1α), A constitutive promoter containing glycerin kinase (PGK) and ubiquitin C (UbC) It is commonly used. CAG promoters are frequently used to promote high levels of gene expression. The powerful synthetic promoter used is the following sequence: (C) cytomegalovirus (CMV) ) Initial enhancer element, (A) promoter of chicken β-actin gene, 1 The exons and first introns of the (G) rabbit β-globin gene, as well as the splice of the (G) rabbit β-globin gene It was built from a Xcepter.

[0094] Furthermore, transcriptional regulators and any additional genetic material may be provided along with the cleavage sequence. Such sequences are sequences that are recognized by entities capable of specifically cleaving DNA. The restriction site is the target sequence of the restriction enzyme, or it is a nuclease, recombinase, or riboza. Includes sequences for recognition by other DNA cleavage entities such as immunoassays or artificial constructs. Each may contain one cleavage sequence, but preferably two or more. These cleavage sequences This allows for the selective removal of selected portions or all of the insert from the GSH. This could be any suitable point within the insert. Therefore, this method involves inserting from the GSH. It can be extended to remove and / or replace an object or part thereof. Therefore, the cutting site may be adjacent to some / all of the inserts that are to be removed. Nodal factors and / or further genetic material can be removed using this method.

[0095] Some of the inserts are up to 99% of the insert, i.e., 1-99%, 90%, 80%, 70%, 60%, 5%. It may be 0%, 40%, 30%, 20%, 10%, or any portion less than 10%.

[0096] The portion of the insert adjacent to the cutting site may preferably contain a constitutive promoter. Alternatively, the constitutive promoter does not contain adjacent regions where the cleavage sequence is located.

[0097] The preferred cutting arrangement allows for direct replacement of the removed insert, and the Cre Recombin This is the loxP site for the enzyme. Alternatively, the cleavage sequence is the lox for the Dre recombinase. It is a part of the body.

[0098] The first GSH insertion occurs at both loci of the genome, so each allele is affected by the insertion. It is preferable that it be modified. This encodes a transcription regulator and any related genetic material. This enables greater expression from genes.

[0099] The second GSH can be any suitable GSH site. Expression of the inserted induction cassette is transcribed. The second GSH site is associated with the endogenous promoter, as if it were only under the control of a regulatory protein. In some cases, it is preferable that it is not kicked.

[0100] The induction cassette contains a desired gene sequence, preferably a DNA sequence to be introduced into cells. The introduction of induction cassettes into the genome involves the addition or embedding of gene sequences that enable gene expression. Knocking down / knocking out the intrinsic expression has the potential to alter the phenotype of the cell. The present invention provides a method for controlling gene sequences (or more) within an induction cassette in cells. Provides a transcription.

[0101] The desired gene sequence for insertion is preferably a DNA sequence that encodes an RNA molecule. The RNA molecule can have any sequence, but preferably it is coding RNA or non-coding RNA. It is RNA. Coding RNA or messenger RNA codes for polypeptide sequences, and The transcription of RNAs like this leads to the expression of intracellular proteins. Non-coding RNAs are functional It can be, and is not limited to, microRNAs, small interfering RNAs, Piwi-interacting RNAs, and Chisense RNA, small nuclear RNA, small nucleolar RNA, small Cajal body RNA, Y RNA, enhancer RNA, guide RNA, ribozyme, small hairpin RNA, small temporal RNA (Small temp oral RNA), trans-acting RNA, small interfering RNA and subgenomic messenger RNA can be obtained. Non-coding RNA can also be known as functional RNA. Some types of RNA are regulated in nature and can downregulate gene expression, for example, by being complementary to a part of the DNA of mRNA or a gene. MicroRNA (miRNA; 21 - 22 nucleotides) is found in eukaryotes, and the miRNA and effector complex of the enzyme can act via RNA interference (RNAi) to cleave complementary mRNA, prevent mRNA from being translated, or promote its degradation. Another type of small interfering RNA of RNA (siRNA; 20 - 25 nucleotides) acts via RNA interference in a manner similar to miRNA. Some miRNAs and siRNAs can reduce or increase the transcription of their target genes by methylating the target genes. Animals have Piwi-interacting RNA (piRNA; 29 - 30 nucleotides), which is active in germline cells and is thought to be a defense against transposons. Many prokaryotes have CRISPR RNA, a regulatory system similar to RNA interference, and such a system includes guide RNA (gRNA). Antisense RNA is widespread, most of which downregulate genes, but some are It can act in this way. Many long non-coded genes regulate eukaryotes. There are ng RNAs, and one such RNA covers one X chromosome in female mammals. Xist is used to deactivate it. Therefore, it is a function that can be used in the method of the present invention. There are many sex RNAs.

[0102] Therefore, the induction cassette may contain a gene sequence that is a protein-coding gene. This gene is either not naturally present in cells, or may be naturally present in cells, but Controllable expression of the gene is required. Alternatively, the induction cassette is particularly for gene control. Mutant versions and modifications of genes present in cells for therapeutic purposes or to derive disease models. It could be version or the correct version. Therefore, the induction cassette is of the same type. Transgenes originating from different organisms (i.e., disease / mutated versions of human-derived genes, young It may contain (or be derived from a human wild-type gene) or from a different species.

[0103] In any aspect or embodiment, the gene sequence contained within the induction cassette is a synthetic arrangement It could be a line.

[0104] The induction cassette provides any suitable gene sequence that is desired to be inserted into the cell's genome. It may include. Therefore, the gene sequence is a gene or mechanism that encodes a protein product. Ribonucleic acid (RNA) with function (nuclear small RNA (snRNA), antisense RNA, microRNA) (miRNA), small interfering RNA (siRNA), transfer RNA (tRNA), and CRISPR-RNA (cr It is transcribed into RNA and other non-coding RNAs (ncRNAs), including guide RNA (gRNA). It could be an array.

[0105] Therefore, the induction cassette is used for any genetic material whose transcription is desired to be controlled within the cell. Genetic sequences may be included. As will be further explained below, the selected gene sequences are cell type and It depends on the intended use of the modified cells.

[0106] For example, in gene therapy, the wild-type gene sequence is used as a component of the induction cassette. It may be desirable to provide it. In this situation, the gene sequence may be provided to any human or These can be protein-coding genes in animals. An example of a protein-coding gene is human β Globin gene, human lipoprotein lipase (LPL) gene, CHM gene and more It contains human Rab escort protein 1 encoded by a gene. Or, the inducer The lead cassette cleaves growth factors and / or propeptides, including BDNF, GDF, NGF, IGF, and FGF. This can lead to the expression of an enzyme that can form an active form. Gene therapy also involves antisense The following types of RNA, miRNA, siRNA, or RNA that interferes with the expression of another gene within a cell are used. This can be achieved by expressing an induction cassette containing the gene sequence that triggers the expression.

[0107] Alternatively, if the cells are stem cells, the induction cassette is, in this specification, a master regulator. It may contain gene sequences that encode important lineage-specific master regulators, abbreviated as "children". Master regulators are transcription factors, transcription regulators, cytokine receptors, or signaling factors. It can be one or more of signaling molecules, etc. The master regulator is the cellular system that expresses it. These are expression genes that influence cell lineage. The network of master regulators is a network of genes that influence cell lineage. may need to be determined. As used herein, a master regulatory gene expressed at the onset of a developing lineage or cell type is involved in lineage specificity by regulating multiple downstream genes either directly or through a cascade of gene expression changes. A master regulator, when expressed, has the ability to respecify the fate of cells that are destined to form other lineages. Examples of master regulators include the myogenic transcription factor MyoD and the hematopoietic transcription factor SCL. In particular, master regulators include nervous system: oligodendrocytes: SOX10, OLIG2, NKX2.2, NKX6.2; astrocytes: NFIA , NFIB, and SOX9; neurons: Ascl1, neurogenin, and NeuroD, Pax6, Neurog 2, Ascl1, Dlx2, and NeuroD1; hematopoietic cells including erythrocytes and megakaryocytes: GATA1, FLI1 and TAL1 mesenchymal lineages: skeletal muscle: MYOD; cardiomyocytes: Gata4, Mef2c, Baf60c and Tbx5; bone: L-Myc (RXOL ) Runx2, osterix, Oct4; cartilage: c-Myc Klf4, SOX9; and brown adipocytes: C / EBP -β and c-Myc are included, but are not limited to these. endoderm pancreatic cell types: PDX1 and GATA6 stem cells: epiblast layer SC: Oct4, Sox2, Klf4 and c-Myc are included, but are not limited to these.

[0108] Alternatively or additionally, the gene sequence or additional genetic material may be a gene whose function needs to be investigated such that the effect of its expression on cells can be examined by controllable expression. The gene may be a growth factor and / or cytokine for which the cells are used in cell transplantation for and / or The gene may contain caine, and / or the gene may be a component of a reporter assay.

[0109] Furthermore, the function of this gene sequence is to knock down the expression of endogenous genes. It encodes non-coding RNA, or encodes non-coding RNA within the cell. It could be a DNA sequence. Alternatively, the gene sequence could result in the knockout of an endogenous gene. It can encode guide RNA for the RISPR-Cas9 system.

[0110] Therefore, the present invention relates to a method for knocking down the expression of endogenous genes within cells. The method is as described above, and the induction cassette is operable to the induction promoter. It includes a gene sequence encoding a non-coding RNA linked to the non-coding RNA, and the non-coding RNA NA suppresses the expression of the endogenous gene. The non-coding RNA is involved in RNA interference and AN Gene expression can be suppressed by any appropriate means, including cysses RNA. Therefore, the gene The gene sequence encodes shRNA that can interfere with the messenger RNA of endogenous genes. obtain.

[0111] The reduction in endogenous gene expression can be partial or complete - that is, expression is non-com Compared to cells before induction of transcription RNA, the values ​​were 50, 55, 65, 70, 75, 80, 85, 90, 91, and 9. The decrease may be 2, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0112] Any other suitable system for gene knockout may be used, but the method of the present invention Furthermore, the CRIPSR-Cas9 system is used to knock out endogenous genes within cells. In this situation, the Cas9 gene is constitutively expressed, and therefore possesses the genes of transcription regulators. It is preferable that it be included in the first GSH. The gRNA coding gene sequence is inserted into the second GSH. It may be included in the induction cassette. The gRNA has a scaffold sequence necessary for Cas9 binding and is modified It is a short synthetic RNA composed of approximately 20 nucleotide target sequences that define the genome target. Therefore, Cas9's genomic targeting is achieved simply by altering the target sequence present in the gRNA. It can be modified. The main use of such systems is to knock out genes. Therefore, the goal is to design gRNAs that target endogenous genes, but selectively activate the target gene. To sexualize or inhibit DNA, purify specific DNA regions, and further modify DNA to enable imaging. It can also be used in all possible applications.

[0113] The induction cassette contains a gene sequence operably linked to an induction promoter. A "romortator" is a nucleotide sequence that initiates and regulates the transcription of polynucleotides. An "inducing promoter" is a promoter that controls the expression of a gene sequence operably linked to it. This invention relates to a nucleotide sequence controlled by precipitates, cofactors, regulatory proteins, etc. In this case, the control is carried out by transcription regulatory proteins. (Term: promoter or) The "control element" includes the full-length promoter region and the functional aspects of these regions (e.g., It is intended to include a segment that controls copying or translation. "Operablely linked" "ta" means that the components described in that way are configured to perform their normal function. This refers to the arrangement of rement. Therefore, a predetermined proment is operably linked to the gene sequence. The enzyme can, if the appropriate enzyme is present, bring about the expression of its sequence. The romor does not need to be adjacent to the sequence as long as it functions to direct the expression of the sequence. Therefore, for example, the intervention of a sequence that is not translated but is transcribed, along with the promoter sequence, It can be present between gene sequences, and the promoter sequence may also "create" the gene sequence. It can be considered as "movably connected". Therefore, "operably connected" The term refers to the process by which the transcription complex recognizes the promoter element, and the transition of the induction cassette. The promoter element and gene sequence within the induction cassette can be arbitrarily selected to enable the start of the copy. It is intended to include the interval or direction.

[0114] Furthermore, other genetic material can also be operably linked to the induction promoter. The genetic material includes genes such as marker or reporter genes, RNA coding sequences, and genetic material. It is possible. Such further genetic material has been described previously. In some situations, It is sometimes desirable for the induction cassette to contain a suicide gene, but the gene sequence itself may be a cancer gene. It must not be a suicide gene for genetic therapy. The suicide gene must be in the same induction cassette. The same induction promoter may be used, or separate induction promoters may be used to allow separate controls. It can be a motor. Such a gene can be a donor / trait if certain conditions are met. This could be useful in gene therapy situations where it is desirable to destroy affected cells. Suicide genes either induce apoptosis in cells or are supplied externally to make them function. It is a gene that expresses a protein that may require a cofactor or co-agent. The agent can be converted into a highly cytotoxic entity by the product of a suicide gene.

[0115] Furthermore, the induction cassette may contain a cleavage sequence. Such a sequence specifically affects DNA A sequence recognized by an entity capable of cleaving it, and a target sequence for restriction enzymes. Restriction sites or other DNs such as nucleases, recombinases, ribozymes or artificial constructs A contains a sequence for recognition by the cleavage entity. It may contain at least one cleavage sequence, but is preferable. There are two or more of these cut sequences. These cut sequences are located in the selected portion of the cassette, or the entire cassette. Any suitable point in the cassette that allows for the selective removal of the body from GSH This may be the case. Therefore, this method removes and / or replaces the GSH from the cassette or a part thereof. Replacement is possible. Therefore, the cleavage site is a gene that can be removed if desired. It may be adjacent to part / all of the row. This method removes the induction cassette and / or further genetic material. It can bring about departure.

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

[0117] The insertion region adjacent to the cleavage site contains a promoter operably linked to the gene sequence. In some cases, this may be preferable. Alternatively, a promoter operably linked to the gene sequence may be used. The cleavage sequence is not included in the adjacent region.

[0118] The preferred cutting arrangement allows for direct replacement of the removed insert, and the Cre Recombin This is the loxP site for the enzyme. Alternatively, the cleavage site is the r site for the Dre recombinase. It could be an ox site.

[0119] Transcriptional regulatory proteins and induction cassettes, along with any relevant genetic material, are involved in the cellular genotherapy. It is inserted into different GSHs within the unit.

[0120] Insertion into GSH is preferably, in particular, within the GSH sequence as described above. Polynucleation into a specific sequence Any suitable technique for the insertion of creotide may be used, some of which are described in the art. The appropriate technique involves introducing a cut at the desired location and rearranging the vector into the gap. This includes any method that makes this possible. Therefore, it is important for target site-specific genome modification. The first step is to create a double-strand DNA break (DSB) at the genomic locus to be modified. The repair mechanisms of separate cells are used to repair DSBs and to introduce desired sequences. These can be used, and these are non-homologous end-joint repairs (NHEJs) that are more prone to errors. and mediated by a donor DNA template that can be used to insert an induction cassette. This is homologous recombination repair (HR).

[0121] Several technologies enable the generation of customized site-specific DSBs within the genome. These are present in the environment. Many of these are zinc finger nucleases (ZFNs) and transcription activators. Child effector nucleases (TALENs) or short palindromes clustered and arranged in a regular pattern Array repeat (clustered regularly interspaced short palindromic repeat) / CRISPR related Customized endonucleases such as the linked protein (CRISPR / Cas9) system (Gaj, T et al., "ZFN, TALEN, and CRISPR / CasV for Genome Genetic Engineering") "ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering" Ends Biotechnol, 31:397-405, July 2013).

[0122] Zinc finger nucleases are formed by combining the nuclease domain of restriction enzyme FokI with zinc finger nucleases. It is an artificial enzyme created by fusing a DNA-binding domain. The latter cuts DNA. Therefore, it has a nonspecific cleavage domain that must be dimerized. This is two Z The FN monomer enables the dimerization of the FokI domain, which is necessary for cleaving DNA. This means that the DNA-binding domain is designed to target any desired genomic sequence. Each of these Cys2His2 zinc fins recognizes three adjacent nucleotides in the target sequence. This is a tandem array of gar. The two binding sites enable optimal dimerization of the FokI domain. To achieve this, it is separated into segments of about 5-7 bp. Therefore, the enzyme can process DNA at specific sites. It can be cleaved, and the target specificity is such that two adjacent DNA bindings can be used to achieve a double-strand break. It increases by ensuring that elephants must arise.

[0123] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factors / nucleases. These are enzymes. They have a DNA cleavage domain (nuclease) with a TAL effector DNA binding domain. It is created by fusing the main component. Transcription activator-like effectors (TALEs) are actually Because it can be engineered to qualitatively bind to any desired DNA sequence, nuclea When combined with -ase, DNA can be cut at specific locations. TAL Effector It is a protein secreted by Xanthomonas bacteria, and its DNA-binding domain is It contains a highly conserved repeat sequence of 33-34 amino acids, with the 12th and 13th amino acids being different. These two positions are highly diverse and have a strong affinity for the recognition of specific nucleotides. This shows a relationship. This direct relationship between amino acid sequences and DNA recognition allows for the application of two variable positions. By selecting a combination of repeat segments containing specific residues, specific DN can be determined. Engineering processing of A-binding domains has become possible. Therefore, each of the TALENs is a single nucleus. It is constructed from an array of 33-35 amino acid modules that target ostide. By selecting an array of sequences, almost any sequence can be targeted. Again, The nuclease used may be FokI or a derivative thereof.

[0124] Three types of CRISPR mechanisms have been identified, and of these, type II is the most studied. CRISPR / Ca The s9 system (Type II) is determined by a short guide RNA using the Cas9 nuclease. It creates double-strand breaks in DNA at specific locations. The CRISPR / Cas system is used for exogenous gene elements. It is a prokaryotic immune system that confers resistance. CRISPR contains short repeats of nucleotide sequences. It is a segment of prokaryotic DNA. Each repeat contains a pre-exposure to an exogenous gene element. A short segment of "protospacer DNA" follows from the dew. The CRISPR spacer is RNA-interfering. Using the interceptor, it recognizes and cleaves exogenous gene elements. The CRISPR immune response involves two... Process: Produced through CRISPR-RNA (crRNA) biosynthesis and crRNA-induced interference. The crRNA molecule is produced by It consists of variable sequences and CRISP repeats transcribed from rotospacer DNA. Each crRNA molecule is a second RNA known as trans-activated CRISPR RNA (tracrRNA). They hybridize, and together these two eventually form a complex with the nuclease Cas9. The portion of the protospacer DNA encoded by crRNA is the protospacer adjacent motif. When adjacent to a short sequence known as PAM, it cleaves the complementary target DNA sequence. This instructs Cas9 to do so. This natural system, among many other applications, is specific to genomic DNA. It has been designed and used to introduce DSB transection at target sites, particularly in the case of streptococcus. A CRIPSR type II system from *Streptococcus pyogenes* may be used. In its simplest form, the CRISPR / Cas9 system is delivered to cells to provide genome editing. It contains two components: Cas9 nuclease itself and low molecular weight guide RNA (gRNA). gRNA is a Cas9 nuclease. A fusion of modified site-specific crRNA (target sequence-oriented) and standardized tracrRNA be.

[0125] After DSB is performed, a donor template homologous to the target gene locus is supplied, and the DSB is correct. It can be repaired by a homologous directional repair (HDR) pathway, which allows for accurate insertion.

[0126] Derivatives of this system are also possible. For example, Ca9D10A, which possesses only nickase activity. A variant of s9 is also available. This means that only one DNA strand is cut, and NHEJ It does not activate. Instead, DNA repair provides a homologous repair template, which enables high fidelity H This occurs only via the DR pathway. (Cas9D10A, Cong L. et al., (2013) Science, 339, 819-82) 3) creates an adjacent DNA nick with two complementary sgRNAs in the adjacent region on the opposite strand of the target site. It is used in a pair of Cas9 complexes designed to do so, which can be particularly advantageous.

[0127] The elements for creating double-strand DNA breaks include plasmids for expression within cells. It can be introduced into the above vectors.

[0128] Therefore, to insert gene / induction cassettes, specifically targeted double strands within the genome are used. Any method for creating the cut can be used in the method of the present invention. The method of inserting the cassette is for ZFN, TALEN and / or CRISPR / Cas9 systems or any of them. It may be preferable to use one or more of the derivatives of the compound.

[0129] After DSB is made by any appropriate means, the gene for insertion is as described below. The offspring / induction cassette may be supplied by any suitable method. The gene / induction cassette and related genes The carrier forms donor DNA for DNA repair in DSBs and uses standard cell repair mechanisms / pathways. It is inserted. The method by which the cutting is initiated is as described above, and which path is used for damage repair. It depends on how it's done.

[0130] The transcription regulatory protein and the induction cassette are placed on separate vectors according to the method of the present invention. It can be supplied for this purpose. A "vector" is a vehicle for artificially transporting genetic material into cells. These are nucleic acid molecules such as DNA molecules that are used for this purpose. The vector is generally an insert (induction). (For example, genes for cassettes or transcription regulatory proteins) and functions as the "skeleton" of vectors. It is a nucleic acid sequence consisting of a larger sequence. The vector can be used as a plasmid, minicircle, or This can be any suitable configuration including linear DNA. The vector is used for gene insertion into the relevant GSH. Along with the minimum sequence to enable input, at least the gene or inducer of a transcription factor Includes an induction cassette operably coupled to the vector. Optionally, the vector may also include, for example, , possessing replication origins (ori) that enable vector amplification within bacteria. Furthermore, or In other words, the vector contains genes for selection markers such as antibiotic resistance genes, and genes for colored markers. It contains a suicide gene.

[0131] Examples of vectors used in the embodiment are shown in Figures 20 to 33.

[0132] The cells used in the method of the present invention may be any human or animal cells. Or rodents such as mice and rats; marsupials such as kangaroos and koalas; bonobos, etc. Non-human primates such as lemurs, gibbons, and apes; camels and llamas Which camels; horses, pigs, cattle, buffalo, bison, goats, sheep, deer, reindeer, ro Domesticated animals such as barnacles, banten, yaks, chickens, ducks, and turkeys; cats, dogs, and rabbits. and mammalian cells such as cells derived from domesticated animals like guinea pigs. The cells are preferably human cells. In certain embodiments, the cells are preferably animal cells. It is of material origin.

[0133] The type of cells used in the method of the present invention is determined at the point when the insertion of genetic material into the GSH site is completed. It depends on the application of cells.

[0134] If the objective is to generate mature cell types from progenitor cells, the cells to be modified are stem cells. Preferably, these are pluripotent stem cells. Pluripotent stem cells can differentiate into almost all cells in the body. They possess the ability. There are several sources of pluripotent stem cells. Embryonic stem cells (ES cells) are early... These are pluripotent stem cells derived from the inner cell mass of a blastocyst, which is a pre-implantation embryo. Induced pluripotent stem cells ( iPSCs express genes and factors that are important for maintaining the definitive characteristics of embryonic stem cells. As a result of being forced into this state, the embryonic stem cells are genetically reprogrammed into an embryonic stem cell-like state. These are somatic cells. In 2006, the introduction of four specific genes encoding transcription factors into adult cells was shown to be effective. It has been shown that these can be converted into pluripotent stem cells (Takahashi, K; Yamanaka, S (2006), Cell). 126 (4): 663-76) However, subsequent research has shown that the number of required genes has decreased / changed. Several members of the Oct-3 / 4 and Sox gene families are latent in the induction process. It was identified as an important transcriptional regulator in the present. Klf family, Myc family, Nanog, Further genes, including some members of LIN28, may improve induction efficiency. Examples of genes that may be included in programming factors include Oct3 / 4, Sox2, Sox1, Sox3, and Sox15. , Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, T cl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 and Glis1 are included. These reprogramming factors can be used individually or in combination of two or more of them. ru.

[0135] The purpose is knockdown or knockdown for further research such as developmental or genetic function research. If the goal is to generate stem cells that have the outgrown gene, the modified cells will be the stem cells. Cells, preferably pluripotent stem cells, or mature cell types. Sources of pluripotent stem cells are As stated above.

[0136] When cells modified by the insertion of an induction cassette are used in a human patient, In some cases, it is preferable that the cells be iPSCs derived from the individual. The purpose is to eliminate the need to adapt the cells to the recipient. Alternatively, WiCell(trademark)(WiC Commercially available iPSCs, such as those available from the ell Research Institute, Inc., Wisconsin, US. It can be used. Alternatively, the cells may be the self or may be provided as tissue-specific stem cells. It can be a cell. Suitable cells include blastocystem cells, induced neural stem cells, and other tissues. It contains specific stem cells.

[0137] In certain embodiments, the cells used are preferably embryonic stem cells or stem cell lines. There are cases where this is not the case. Numerous embryonic stem cell lines are now available, for example, WA01(H1) and WA 09(H9) can be obtained from WiCell, and KhES-1, KhES-2, and KhES-3 are available from Kyoto University. It can be obtained from the Institute of Biomedical Sciences (Kyoto, Japan).

[0138] Since such technology is readily available, embryonic stem cells are particularly useful when the cells are human. In some cases, it may be preferable to extract the embryo without destroying it (Chung, Young et al.). References, Cell Stem Cell, Vol. 2, Issue 2, 113-117). Stem cell lines derived without destroying embryos are also useful. It is applicable. In one embodiment, the present invention does not extend to methods that involve the destruction of human embryos.

[0139] A preferred embodiment of the present invention involves forward programming of pluripotent stem cells to mature cell types. Therefore, the method of the present invention can be used for the production of mature cell types from pluripotent stem cells. This is possible. As described above, in this embodiment of the present invention, the induction cassette for insertion into the second GSH The set is preferably one or more master regulators. These induction cassettes are cell This could allow it to be programmed into a specific lineage, and different induction cassettes can lead to mature cell types. Used to direct the differentiation of nerve cells, muscle cells, bone cells, chondrocytes, epithelial cells, Any species of mature cell including, but not limited to, secretory cells and / or blood cells. A similar type is planned.

[0140] The inventors of this application have developed a rapid, efficient and effective method for generating substantially any mature cell type. We developed a scalable method. Such a simple, inexpensive method is particularly useful for regenerative medicine. It has a certain value. Previous forward programming techniques used the Tet-On system. Attempts to include all substances in a single vector / site (all-in-one Tet-On), or the inducement The guide cassette is inserted into one AAVS1 allele, and the control system is inserted into the other AAVS1 allele. Attempts have been made to introduce this (DeKelver et al., 2010, Genome Res., 20, 1133-43 and Qian et al.). (Reference, 2014, Stem Cells, 32, 1230-8). Surprisingly, it was developed and described herein. The dual GSH targeting method has many unexpected advantages. The gene inserted into the first GSH and Voltage promoter interference exists between the gene sequence of the induction cassette inserted into the second GSH. No. Secondly, because there is less substance to be inserted into each site, the amount from the vector is smaller. This enables the insertion of large cargo. Thirdly, this method maximizes the number of copies that can be safely inserted. Yes. Fourth, this method allows for greater design flexibility. Finally, this method is repo This allows for the insertion of additional genetic material, including ter genes and miRNA switches. The present invention provides a robust and efficient method for producing mature cells from pluripotent cells. This has been proven.

[0141] The gene is inserted into the first GSH, and the induction cassette containing the transgene is inserted into the second GSH. Pluripotent stem cells are cultured to allow forward programming to occur. These culture conditions may be specific to the type of pluripotent stem cells used, or This may depend on the final mature cell type. Regardless of the culture conditions used, exogenous substances may induce It is continuously supplied to control the expression of gene sequences within the lead cassette and induce transcription, It is removed afterwards, or, as mentioned above, depending on its mode of action, when transcription is required. It can be supplied to.

[0142] If the goal is to program stem cells, then the induction of a master regulator is Providing the cells with detailed extracellular stimuli is advantageous in order to support differentiation along with supplying the cells with a set of stimuli. This is possible. Cellular reprogramming strategies involve extracellular signaling cues and master regulators. This can be improved by combining the overexpression of the child or transcription factor. This modulates the major signaling cascades involved in the development of that particular mature cell type. This may make it possible to systematically screen for differentiation-promoting factors. An example of this is shown in Example 3.

[0143] In one embodiment, the present invention is a method for generating muscle cells from pluripotent stem cells, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) A MYOD operably linked to the inducible promoter at the second gene safe harbor site. A step of targeted insertion of a gene, wherein the inducing promoter is transmitted to the transcription regulatory protein. The above process is thus adjusted, It includes, and the first and second gene-safe harbor sites are different, The present invention provides a method comprising culturing the cells in the presence of retinoic acid.

[0144] The MYOD1 gene is a myogenesis differentiation 1 protein-coding gene. Preferably, retinoin The acid (RA) is all-trans RA.

[0145] In another embodiment, the present invention relates to a method for generating muscle cells from pluripotent stem cells expressing MYOD1. The present invention provides a method comprising culturing the cells in the presence of retinoic acid. .

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

[0147] In a further aspect, the present invention relates to oligodendrocyte myocytes from pluripotent stem cells. A method of generation, a) Targeted insertion of a transcriptional regulatory protein-coding gene into the first gene-safe harbor site. Process; and b) SOX operably linked to the inducible promoter at the second gene safe harbor site A step of targeted insertion of 10 genes, wherein the inducing promoter is transmitted to the transcription regulatory protein The above process is thus adjusted, It includes, and the first and second gene-safe harbor sites are different, The present invention provides a method comprising culturing the cells in the presence of retinoic acid.

[0148] The cells used for this purpose may be animal or human cells. If the cells are animal cells... Preferably, the animal is a domesticated animal as previously defined.

[0149] The SOX 10 gene encodes the transcription factor SOX 10. Preferably, retinoic acid (RA) is used. This is Lutrans RA.

[0150] The cells used in the method of the present invention are pluripotent, and the resulting cells are master-regulated. By expressing the factor, specific stem cells, progenitor cells, or cells with desired properties can be expressed. They may be mature cells. These lineage-specific stem cells, progenitor cells, or mature cells may be any appropriate It can be used in various ways. For example, the mature cells can be introduced into the body of a human or animal, depending on the cell type. It can be used directly for transplantation. Alternatively, the cells can be used to enhance the effects of drugs on gene expression. Cells for research can be used as test material for studies involving the interaction of specific genes with drugs. To investigate the controllable expression of that gene sequence, inducers possessing gene sequences with unknown function are used. It may involve the use of a guide cassette. Furthermore, it may involve growth factors or cytokines, etc. This could allow the cells to be used to produce large quantities of the desired material.

[0151] In a different embodiment, the cells may be used in tissue engineering. Tissue engineering involves human or This requires the creation of tissues that can be used to replace animal tissues or even entire organs. The method of study is known to those skilled in the art, but the foot to which cells are applied to produce tissue / organs This includes the use of a field (extracellular matrix). These methods involve "artificial" trachea, bladder, and liver. , pancreas, stomach, intestines, blood vessels, heart tissue, bone, bone marrow, mucosal tissue, nerves, muscles, skin, kidneys or palpable tissue It can be used to produce other tissues or organs. Method for producing tissue. Another method involves directly printing cells to create tissues in three dimensions (3D). This may include additive manufacturing, also known as printing. Therefore, the present invention may be an optional invention. As described in the embodiment, a method for producing tissue using generated cells is provided.

[0152] Tissue produced using cells created according to the method of the present invention is a human or animal body It can be used for transplantation into the body. Alternatively, if the cells are of animal origin, the tissue may be used. It can be used in in vitro / cultured meat. The main cell type of cultured meat is muscle cells. However Such tissues involve the use of combinations of cell types prepared according to the method of the present invention. It is possible. These include muscle cells, blood vessel cells, blood cells, and fat cells. (Cells) may be. If the purpose of the modified tissue is for cultured meat, the cells may be livestock animals. It can be extracted from.

[0153] The methods of the present invention are also used in research, gene therapy including gene vaccines, and in vitro disease models. For various reasons, including the generation of non-human in vivo models, pluripotent stem cells are not This may also be performed in cells.

[0154] Therefore, the cells used in the method of the present invention may be any type of adult stem cell. These are undifferentiated cells that can develop into many, but not all, cell types. Adult stem cells are found throughout the body and divide to replace dead cells and regenerate damaged tissue. These are undifferentiated cells. As they are also known as somatic stem cells, they are not pluripotent. Adult stem cells are found in the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestines, liver, and ovarian epithelium. It has been identified in many organs and tissues, including the testes. It labels somatic stem cells. Therefore, those skilled in the art will recognize that a single adult stem cell can give rise to all appropriate differentiated cell types of tissue. It must be demonstrated that identical cell lines can be genetically reproduced. (Presumed adult stem) To experimentally confirm that the cells are indeed stem cells, the cells were subjected to the following conditions in the culture medium: To produce genetically identical cells, or to give a purified population of these cells to an animal The tissue must be rearranged after transplantation. Appropriate cell types include nerve, mesenchymal, and endodermal cells. This includes, but is not limited to, stem cells and progenitor cells.

[0155] Alternatively, the cells used may be of the mature cell type. Such cells undergo differentiation and specialization. They are unable to develop into different cell types. Mature cell types include nerve cells and muscle cells. This includes, but also includes, osteocytes, chondrocytes, epithelial cells, secretory cells, and / or blood cells. Not limited to these. Mature cell types can be any cells derived from the body of a human or animal.

[0156] Somatic stem cells and mature cell types are modified in accordance with the present invention and then used for gene therapy or It can be used in applications such as gene vaccination. Gene therapy involves targeting the cell nucleus for therapeutic purposes. It can be defined as the intentional insertion of foreign DNA. Such a definition would involve the wild-type defect of the gene. Provision of genes or multiple genes to cells to provide a version, expression of target genes Addition of genes for RNA molecules that interfere with (which may be defects), suicide genes (harmless prodrugs) The enzyme that converts ganciclovir (GCV) into a cytotoxic drug in herpes simplex virus thyroid Din kinase (HSV-tk) and cytosine deaminase (CD), etc., for immunotherapy or cancer treatment. The provision of DNA vaccines (including adoptive immunotherapy) and any other use of cells for therapeutic purposes. This includes gene donation.

[0157] Typically, the method of the present invention is particularly useful in DNA vaccines for intracellular transcription, and is preferred It can be used to insert desired gene sequences for expression. DNA vaccines are typically This encodes a modified version of the infectious organism's DNA. DNA vaccines are selected for use against infectious organisms. When administered to a target that expresses a protein, it typically triggers an immune response to the protein, which is usually protective. Let's begin the answer. DNA vaccines also call tumor antigens in cancer immunotherapy approaches. It is possible.

[0158] DNA vaccines are used for cancer, allergies, toxicity, and, but are not limited to, fungi, and human papillomavirus (HPV). Roman virus (HPV), HIV, HSV2 / HSV1, influenza virus (types A, B and C), Liovirus, RSV virus, rhinovirus, rotavirus, hepatitis A virus, measles virus Viruses, parainfluenza viruses, mumps viruses, varicella-zoster virus, rhino Tomegalovirus, Epstein-Barr virus, adenovirus, rubella virus, human T14 virus Hepatitis V1 virus (HTLV-I), hepatitis B virus (HBV), and hepatitis C virus (HCV) are causes of cellular lymphoma. V) Includes hepatitis D virus, poxvirus, Zika virus, Marburg virus, and Ebola. Viruses; bacteria including Neisseria meningitidis and Haemophilus influenzae (type B); and parasitic diseases This includes, but is not limited to, infections caused by pathogens such as natrioxal compounds, and the treatment of several other conditions. It may contain nucleic acid sequences that code for antigens for prevention. DNA vaccines can be used for any appropriate disease. It may contain nucleic acid sequences encoding antigens derived from the original organism. These antigens are involved in diseases in humans or animals. It originates from pathogens, and in particular, it may originate from viral pathogens.

[0159] The DNA vaccine inserted into the GSH may also contain nucleic acid sequences encoding tumor antigens. Examples of ulcer-associated antigens include members of the MAGE family (MAGE 1, 2, 3, etc.), NY-ESO-I, and SSX- Cancer antigens such as 2, differentiation antigens such as tyrosinase, gplOO, PSA, Her-2 and CEA, and mutant autoantibodies This includes viral tumor antigens such as E6 and / or E7 derived from proto- and oncogenic HPV types, but these This is not limited to, however. Further examples of specific tumor antigens include MART-I, Melan-A, p97, beta-HCG, GaINAc, MAGE-I, MAGE-2, MAGE-4, MAGE-12, MUCl, MUC2, MUC3, MUC4, MUC18, CEA, DDC , PlA, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, Kl 9, Tyrl, Tyr2, Members of the pMel 17 gene family, c-Met, PSM (prostatic mucin antigen), PSMA (prostatic mucin antigen) (Specific membrane antigen), prostatic secretory protein, α-fetoprotein, CA 125, CA 19.9, TAG- 72 contains BRCA-I and BRCA-2 antigens.

[0160] The inserted gene sequence may generate other types of therapeutic DNA molecules. For example, DNA molecules are genetic diseases caused by a dysfunctional version of the gene in which the target is located. It can be used to express functional genes that are affected by disease. Examples of such diseases include This includes Duchenne muscular dystrophy, cystic fibrosis, Gaucher disease, and adenosine degeneration. This includes ADA deficiency. Other diseases for which gene therapy may be useful include AIDS. Cancer, neurological disorders, cardiovascular diseases, hypercholesterolemia, various types of anemia, thalassemia and hemophilia , and inflammatory diseases, autoimmune diseases, chronic diseases, including various blood disorders such as emphysema. This includes diseases and infectious diseases. For the treatment of solid tumors, toxic peptides (i.e., Genes encoding chemotherapeutic agents such as lysine, diphtheria toxin, and cobra venom factor, p53 These genes encode mRNA sequences that are antisense for tumor suppressor genes and transforming oncogenes. Antitumor peptides such as genes, tumor necrosis factor (TNF), and other cytokines, or form Transdominant-negative variants of conversion oncogenes may be expressed.

[0161] Other types of therapeutic DNA molecules are also being considered. For example, active non-coding RNA types The DNA molecules to be transcribed, such as small interfering RNAs (siRNAs), can be inserted. Therefore, The method of the invention involves knocking down the expression of endogenous genes or non-coating them within an induction cassette. This involves methods for knocking out endogenous genes using Ding RNA.

[0162] Therefore, the method of the present invention provides a controllable transferable induction cassette that is specific and stable within it. It can be used to qualitatively insert gene sequences. This is applicable to somatic stem cells and mature cell types. It has numerous advantages in that it ensures that important genes are not destroyed and by-products A more strictly controlled system that can stop the induction cassette from functioning if necessary. It enables gene therapy approaches. It also allows for the study of gene function and development. This enables the knockdown or knockout of tightly regulated endogenous genes.

[0163] The present invention extends to cells produced by the method of the present invention. These cells are transcription regulatory proteins. A first genome-safe harbor site for including and regulated by the transcription regulatory protein A second gene sequence containing a gene sequence operably linked to an inductive promoter. It can be defined as being modified at the Fuhabar site. The two GSHs are different and separate. Preferably, the cell is homozygous at both insertion sites. All elements are as described above. That is correct.

[0164] Cells produced according to any of the methods of the present invention may be applied in diagnostic and therapeutic methods. These cells are used to study cell development, provide a testing system for new drugs, and screen To enable the development of diagnostic methods, to refine treatment plans, and to provide diagnostic tests, etc. It can be used in vitro. These uses constitute part of the present invention. Alternatively, the cells can be used for diagnostic purposes. It may be transplanted into human or animal patients for diagnoses or therapeutic purposes. Use of cells during therapy. This is also included in the present invention. The cells are allogeneic (i.e., removed, modified and in the same individual). The cells may be derived from the returned mature cells or from a donor (including stem cell lines).

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

[0166] array AAVS1 - NCBI GenBank S51329.1 Sequence ID 1: Tet02 19n sequence Sequence ID 2: hROSA insertion site genome sequence Sequence ID 3: STDtetR-nls (nucleotide) and Sequence ID 4: STDtetR-nls (amino acid) Sequence ID 5: OPTtetR-nls (nucleotide) and Sequence ID 6: OPTtetR-nls (amino acid) Sequence IDs 7-80: Primers from Table 3 Sequence ID 81: Figure 18B AAVS1 FWD; Sequence ID 82: Figure 18B AAVS1 REV Sequence ID 83: Figure 18B Tracer FWD; Sequence ID 84: Figure 18B Tracer REV Sequence ID 85: Figure 19E HI POL3 FWD; Sequence ID 82: Figure 19E HI POL3 REV

[0167] This is the genomic sequence of the hROSA26 insertion site. It is the 5' homology arm, the cleavage site ( (Bold), and including the 3' homology arm: (Sequence ID 2). [ka]

[0168] STDtetR-nls (SEQ ID NOs: 3 and 4) Tetracycline-sensitive receptor containing N-terminal SV40 nuclear localization signal (nls, highlighted in gray) Nucleotide and amino acid sequences of the sex-inhibiting protein (tetR). Before or after codon optimization. Reports one of the following sequences (STDtetR and OPTtetR, respectively). A dot indicates that OPTtetR has been introduced. This shows a synonymous mutation. [ka]

[0169] Optimized tetR: Sequence of OPTtetR-nls (sequences 5 and 6) [ka]

[0170] The present invention will now be described in relation to the following non-limiting embodiments.

[0171] Examples Materials and methods used in the examples: Maintenance culture and germ layer differentiation of hPSCs hESC (H9 strain; WiCell) and hiPSC (Cheung et al., Nat.) without feeders or serum. We cultured the cells in Biotechnol. 30, 165-173 (2012). To put it simply, we cultured the cells in gelatin. Seeds were seeded on a culture dish coated with Chin / MEF medium. [MEF medium is Advanced DMEM / F12 (90 %, Gibco), fetal bovine serum (10%, Gibco), L-glutamine (1 mM, Gibco), 2-mercapsule Toethanol (0.1 mM, Sigma-Aldrich) and penicillin / streptomycin (1%, Gibco) [consisted of], supplemented with 10 ng / ml activin A and 12 ng / ml FGF2, chemically defined Prepared culture media [CDM, IMDM (50%, Gibco), F12 (50%, Gibco), concentrated lipids (100x, Gibco), mono Thioglycerol (450 μM, Sigma-Aldrich), insulin (7 μg / ml, Roche), trans Ferrin (15 μg / ml, Roche), bovine serum albumin fraction V (5 mg / ml), and penicillin Cells were cultured in phosphorus / streptomycin (1%). Collagenase was administered every 5-6 days. It was used and subcultured in small clumps.

[0172] Previously published targeted differentiation protocols for endoderm, lateral plate mesoderm, and neuroectoderm. According to the literature by Touboul, T. et al., He patology 51, 1754-1765 (2010), the literature of Cheung et al., (2012) and the literature of Douvaras, P. et al., St (Cell Reports 3, 250-259 (2014)). In short, endoderm of the embryo thorax is affected by FGF2 (20 ng / ml). ), activin A (100 ng / ml), BMP4 (10 ng / ml), Marko Hyvonen, Department of Chemistry, University of Cambridge ( Dept. of Biochemistry, University of Cambridge, and LY-294002 (10 μM, Promega) Obtained by culturing hPSCs for 3 days in supplemented CDM-PVA (without insulin). 3. Neuronal For ectoderm induction, SB-431542 (10 μM, Tocris), LDN-193189 (0.1 μM, Tocris), and RA were used. hPSCs were cultured for 6 days in CDM-BSA supplemented with (0.1 μM, Sigma). 4. Lateral plate mesoderm was treated with FGF2( hPSCs were tested for 36 hours in CDMPVA supplemented with 20 ng / ml, 10 ng / ml BMP4 (R&D), and LY294002 (10 μM). The cells were cultured for 3.5 days, and then cultured again in CDM-PVA supplemented with FGF2 (20 ng / ml) and BMP4 (50 ng / ml). It was obtained by doing so.

[0173] hESC differentiation Differentiation was initiated in the adherent culture of hESCs at 48 hours after passage. Medium changes were performed as usual. This was performed daily, and the amount was adjusted relative to the cell density. Mature cell types were previously described in the art. The obtained mature cell types included nerve cells, osteocytes, chondrocytes, and smooth muscle cells. This included cardiac fibroblasts, cardiomyocytes, intestines, pancreas, hepatocytes, bile ducts, or lungs.

[0174] Gene targeting constructs and molecular cloning The design and construction of hROSA26 gRNA and Cas9n expression plasmids are described herein as: hROSA26 gene CRISPR / Cas9n for specifically targeting loci and inserting induction cassettes using homologous recombination. This is described in the strategy based on CRISP to induce genomic DSBs at the correct integration site. We designed an R / Cas9 nickase system that is guided to its genomic target site by a single gRNA. In contrast to the commonly used wild-type Cas9 nuclease, the D10A mutant Cas9 nickasase ( Cas9n uses a properly designed pair of gRNAs to create single-strand breaks on both strands of target DNA. It is sometimes instructed to be implemented. This strategy effectively doubles the number of bases required for genome editing. By increasing it, specificity is increased. Web-based software "CRISPR design tool Using "Lu", potential target sites for crRNA-inducible nucleases close to the integration site are identified. Defined: A 250 bp sequence stretch around the target site (12 bp on each site of the actual integration site). Within 5 bp), the top hits were clustered at 97 "high quality" scores where the targeting effect was not predicted. It was a pair of gRNAs that had reached the target level. [ka] The PAM region (in parentheses) was newly synthesized and ligated into the expression vector. Each plasmid encodes one of two gRNAs and the Cas9n D10A variant (Figure 2). 0 and 21).

[0175] Donor DNA that functions as template DNA to promote homology-directed repair of Cas9n-induced DSBs Sumido was constructed. Two hROSA26 homology arms were fabricated by high-fidelity PCR amplification. 9 Genomic DNA isolated from hESCs served as a template. The 5' and 3' homology arms were respectively The lengths were 904 bp and 869 bp. Both were then multicloned into pUC19 vectors. It was inserted into the ing site. To target the hROSA26 gene locus, two gRs of the plasmid were used to target the cells. Transfected with NA / Cas9n constructs and EGFP donor plasmids (Figure 22).

[0176] The pR26_CAG-rtTA targeting vector (Figure 23) was PCR amplified from the third-generation rtTA (pLVX-Tet3G). The coding sequence of ) was cloned into the BamHI / MluI region of pR26_CAG-EGFP, and therefore, the EGFP distribution The AAVS1 ZFN expression plasmid was constructed by replacing columns. Dr. Kosuke Yusa (Wel It was a generous gift from lcome-Trust Sanger Institute. Inducible EGFP AAVS1-targeted vector The tar was constructed by Gibson Assembly (New England Biolabs), and it contains three insertions The substance is the EcoRI / Hin multicloning site of the pUC19 vector (Thermo Fisher Scientific). It was ligated at the dIII site. The first insert had an AAVS1 homology arm upstream, and a sp Rice acceptor, T2A site and puromycin-resistant cassette (by Rudolf Jaenisch) It contained pTRE-EGFP (addgene 22074, which was PCR-amplified) that was entrusted to us. Second insertion It contained an inducible TRE3G promoter (PCR amplified from pLVX-TRE3G). Third insertion The contents include an EGFP expression cassette and an AAVS1 downstream homology arm (deposited by Rudolf Jaenisch). It contained pTRE-EGFP (amplified by PCR from addgene 22074). The resulting plasmid was This was named pAAV_TRE-EGFP (Figure 32). The NGN2 and MYOD1 coding sequences are respectively (NGN2: Oliver PCR amplified from pLVX-TRE-NGN2, a gift from Brustle; MYOD1: Commercially available cDNA plasmid (Op PCR from en Biosystems MHS6278-202832821, Accession: BC064493, Clone ID: 5022419) The amplified EGFP sequence was cloned into the SpeI / EcoRI region of pAAV_TRE-EGFP, and therefore into the EGFP sequence. By substituting, the pAAV_TRE-NGN2 and pAAV_TRE-MYOD1 (Figure 33) targeting vectors are constructed. It was built.

[0177] Further plasmids were prepared using the same method, and all plasmids used are shown in Figure 20- As shown in 33, these plasmids were either constructed or generously donated. The plasmids used in the examples were (in the order of Figures 20-33), pSpCas9n(BB),_R26-R, pSpCas 9n(BB) (These two plasmid combinations are found on chromosome 3, THUMPDS3-AS1 (ROSA26 It induces specific double-strand breaks in the intron between exon 1 and exon 2 of the constellation Dendrobium. (It is predicted that) R26-L pR26_CAG_EGFP, pR26_CAG_rtTA, pZFN-AAVS1-L-ELD ( Zinc finger nuclease (left), pZFN-AAVS1-R-KKR (zinc finger nuclease (right)), p AAV_CAG_EGFP (donor), pR26-Neo_CAG-OPTtetR (hROSA26 targeting of codon-optimized tetR), pAA V-Puro_iKD (AAVS1 targeting of inducible shRNA), pAAV-Neo_CAG-Cas9 (AAVS1 targeting of Cas9), pA AV-Puro_siKO (AAVS1 targeting of inducible gRNA), pAAV-Puro_siKO-2TO (AAVS1 targeting of inducible gRNA) (A version with two tet operons in the promoter), pAAV_TRE-EGFP (EGFP induction) This includes sexual overexpression (attached) and pAAV_TRE-MYOD1 (MYOD1-induced overexpression for muscle).

[0178] Genetic targeting Targeting of the hROSA26 locus and AAVS1 for gene knockdown and knockout. This was performed by nucleofection. Human pluripotent stem cells (PSCs) were treated with TrypLE Select (Gi Dissociate into single cells using bco) and 2 × 10⁻¹⁶ 6 Individual cells, Lonza P3 primary cells 4D nucleophen Tar X Kit (Lonza P3 Primary Cell 4D-Nucleofector X Kit) and Lonza 4D Nucleofector Nucleofected using the CA-137 cycle of the Nucleofector System. (100 μl reaction volume; total 12 μg of DNA, to be used with two gRNA / Cas9n plasmids and a targeting vector) (Distributed equally). Nucleofected hPSCs were irradiated into multidrug-resistant (DR4) mice. KSR cultures were cultured on germinal fibroblasts and supplemented with FGF2 (4 ng / ml, Department of Chemistry, University of Cambridge). [Advanced DMEM / F12 (80%), knockout serum substitute (20%, Gibco), L-glutamic acid] Min (1 mM), 2-mercaptoethanol (0.1 mM) and penicillin / streptomycin (1 mM) The cells were cultured in a mixture consisting of %). Y-27632 (5 μM, Tocris) was administered 24 hours before and after nucleofection. It was added intermittently to promote cell survival. After 3-6 days, neomycin-resistant hPSCs were treated with G41 for 7-10 days. Selection was performed by adding 8 (50 μg / ml, Sigma-Aldrich). Then, individual clones were selected. Samples were collected, grown under conditions without feeders, and finally analyzed by genotyping. .

[0179] Targeting of the AAVS1 gene locus was also performed by lipofection. Human PSCs were subjected to 6 wells. In the field, seeds were sown under conditions without a feeder, and transfected 48 hours after subculturing. Transfection was performed using lipofectamine 2000 (10 μl / well, Thermo Fisher S). In Opti-MEM (Gibco) supplemented with scientific (2 AAVS1 ZFN plasmids), a total of 4 μg of DNA (2 AAVS1 ZFN plasmids) was added. The drug was evenly distributed to the targeting vector and administered for 24 hours. After 3-5 days, resistant hPSCs were treated with 5-8 The selection was performed by adding puromycin (1 μg / ml, Sigma-Aldrich) for several days. Afterward, individual clones were collected, propagated, and analyzed by genotyping. Antibiotic resistance This can be used to select clonal strains.

[0180] We will verify the integration of drug-resistant hPSC clones from targeted experiments into site-directed induction cassettes. To screen for this purpose, we use genomic PCR to determine the number of target alleles, and if the target is missed, The incorporated DNA was excluded. PCR was performed using LongAmp Taq DNA polymerase (New England Biolabs). This was done using the following methods. Table 2 shows the primer combinations used for various targeting vectors. Report the results. Summarize the results of all targeting experiments in Table 1. Karyotype analysis will be performed using standard G-banding. Performed using the endodontic technique (Medical Genetics Service, University of Cambridge Hospital) Genetics Service, Cambridge University Hospitals. Targeted human PS for chromosome analysis. To prepare C, cells were treated with Y-27632 (5 μM, Tocris) and KaryoMAX colcemid. The cells were incubated at +37°C for 4 hours in fresh culture medium supplemented with (100 ng / ml, Gibco). Subsequently, the cells were harvested as single cells, washed, and pelletized. They were then subjected to hypotonic 0.0 for 5-10 minutes. Treatment with a 55M KCl solution resulted in nuclear expansion and chromosome spreading. Finally, The cells were fixed with methanol and glacial acetic acid (ratio 3:1).

[0181] For OPTiKD, AAVS1 targeting was performed by lipofection as described above. To explain, everything follows the manufacturer's instructions, hPSC, 6W (excluding feeder) Seeds were seeded in a cell plate, and lipofectamine 2000 in Opti-MEM medium (Gibco) was added to each well. Using μl, 48 hours after cell passage, 4 μg of DNA was transfected for 24 hours (2 (The AAVS1 ZFN plasmid and targeting vector were equally divided.) After 4 days, 1 μg / ml pure Romycin was added to the culture medium, individual clones were collected, and they were grown 7-10 days after selection. .

[0182] For single-site OPTiKO, AAVS1 was targeted by nucleofection. Then, following the manufacturer's instructions, the hESC was pretreated with 10 μM Y-27632 (Tocris) for 16 hours. Using Accutase (Gibco), the cells were dissociated into aggregates of 2-8 cells, resulting in 2 × 10⁶ cells. 6 Individual cells, Lonza P3 The original Cell 4D Nucleofector X Kit and Lonza 4D Nucleofector System Using Kuru CA-137, a total of 12 μg of DNA was added per 100 μl (4 μg each for the two ZFN plasmids). The two targeting vectors were each nucleofected with 2 μg. hESCs were irradiated into DR4 (puromycin and neomycin-resistant) mouse embryonic fibroblasts. Seeds were seeded on a feeder layer of cells and cultured in KSR medium supplemented with 4 ng / ml FGF2 and 10 μM Y-27632. Nurturing (only this last part for the first 24 hours). Four days later, puromycin and neoma hPSC colonies possessing both isin resistance genes were treated with 25 μg / ml-1 Geneticin (G418 sulfur). Selection was performed using salts (Gibco) and 0.5 μg / ml-1 puromycin for 7-10 days. Subsequently, individual Clones were collected and propagated under conditions without feeders.

[0183] hE of AAVS1-EGFP, ROSA26-EGFP, ROSA26-STDtetR, ROSA26-OPTtetR, and ROSA26-EGFPd2 SC is lipoferised using a targeting vector having AAVS1 ZFN or ROSA26 CRISPR / Cas9n pair (as described above). Created by nucleofection (AAVS1 locus) or nucleofection (ROSA26 locus). 2 μg / ml-1 blastosidine S-HCl (Gibco) was administered to the pR26-Bsd_CAG-EGFPd2 plasmid. It was used for the purpose of inducible EGFP overload in individuals possessing ROSA26-rtTA and AAVS1-TRE-EGFP transgenes. The method for creating expression hESCs is described elsewhere. In short, it involves creating cells... Initially, nucleofection of pR26-Neo_CAG-rtTA with the ROSA26 CRISPR / Cas9n plasmid was performed. Next, the pAAVPuro_TRE-EGFP lipofection is performed using the AAVS1 ZFN plasmid. Next, we targeted the gene.

[0184] Gene-targeted hPSC clones were screened by genomic PCR, and site-specific targets were identified. We confirmed the targeting, determined the number of alleles to be targeted, and identified cases where the targeting plasmid missed its target. The embedded system was excluded (see Figure 16A).

[0185] Overexpression of induction cassette Overexpression of induction cassettes (EGFP, NGN2, MYOD1, and OLIG2-SOX10, respectively) in culture medium. This was induced by the addition of doxycycline hydrate (Sigma-Aldrich). (See specific description.) Unless otherwise specified, doxycycline was used at a final concentration of 1 μg / ml. (Contains doxycycline) The culture medium was changed every 24 hours, protected from light. EGFP-expressing cells were used as described herein. Those that express NGN2 are called OPTi-EGFP, those that express NGN2 are called OPTi-NGN2, and MYOD1-expressing cells are called OPTi-MYOD We refer to it as 1, and the OLIG2-SOX10 expressing cells as OPTi-OLIG2-SOX10.

[0186] Inducible gene knockout and knockdown Unless otherwise stated in the figure caption or examples, tetracycline hydrochloride (Sigma-Aldric h) was used at a concentration of 1 μg / ml to induce gene knockdown or knockout. Neurons Induction. Pluripotent OPTi-NGN2 cells were dissociated into single cells using TrypLE and placed in a 12-well plate. Matrigel (35 μg / cm³) with a cell density of 75,000 cells per unit. 2 , Scientific Laboratory Supplies Seeds were sown on coated trays. Forward programming was performed 24-48 hours after division. It started with. Unless otherwise specified, glutamax (100x, Gibco), non-essential amino acids ( 100x dilution, Gibco), 2-mercaptoethanol (50 μM), penicillin / streptomycin ( Induction was performed in DMEM / F12 (Gibco) supplemented with 1% and doxycycline (1 μg / ml). Two days after induction, the culture medium was treated with glutamax (100x), B27 (50x, Gibco), and BDNF (10ng). / ml, Peprotech), NT3 (10ng / ml, R&D Systems), penicillin / streptomycin (1 The patient switched to Neurobasal medium supplemented with %, and doxycycline (1 μg / ml).

[0187] Induction of skeletal muscle cells Pluripotent OPTi-MYOD1 cells were dissociated into single cells using TrypLE and placed in one well of a 12-well plate. Cells were seeded at a density of 100,000 cells on gelatin / MEF medium-coated dishes. Programming was started 24-48 hours after the split. Unless otherwise specified, L-glutamic acid Min (2 mM), 2-mercaptoethanol (50 μM), penicillin / streptomycin (1%) ), insulin (7 μg / ml), all-trans retinoic acid (1 μM, Sigma-Aldrich), and Induction was performed in DMEM (Sigma-Aldrich) supplemented with doxycycline (1 μg / ml). Five days after induction, the culture medium was supplemented with CHIR99021 (3 μM, Tocris) and heat-inactivated horse serum (2%, Gibco). It was enriched and its maturity was strengthened.

[0188] Induction of oligodendrocytes Pluripotent OLIG2-2A-SOX10 OPTi-OX hPSCs were cultured on gelatin / MEF-coated incubators. They were propagated using Knee. Before induction began, they were treated with SB and LDN overnight. The next day, Doki Induction was initiated in CDM supplemented with cycycline (1 μg / ml) and RA (0.1 μM). On day [number], PDL / laminin-coated culture dishes (100,000 cells per well of a 12-well plate) On the cells, RA (0.1 μM), PM (1 μM), and Y-27632 (5 μM), PDGFaa (20 ng / ml, Peprote Cells were divided in CDM supplemented with ch), FGF2 (5 ng / ml). The next day, the cells were treated with glutamax. (100x dilution), non-essential amino acids (100x dilution), 2-mercaptoethanol (1000x dilution), penicillin- Streptomycin (100x dilution), N2 supplement (100x dilution), B27 supplement (50x dilution), Insulin 7 μg / ml (Marko Hyvonnen), T3 60 ng / ml (Sigma), Biotin 100 ng / ml (Sigma), d The oligodendrocyte medium was switched to DMEM / F12 supplemented with 1 μM b-cAMP (Sigma). In oligodendrocyte medium, dox (1 μg / ml), PDGFaa (20 ng / ml), FGF2 (5 ng / ml), RA (0. RA (1 μM) and PM (1 μM) were supplemented. Seven days after induction, RA and PM were removed. In the proliferative state, the induced cells were observed. To maintain the cells, the cells are continuously conditioned every four days in the presence of mitogens PDGFaa and FGF2. (75,000 cells per well in a 24-well plate). Proliferative oligodendrosa PDGFaa and FGF2 were removed for differentiation of the ion precursor. Human recombinant NT3 (5 ng / μl, R&D (Systems) were added to enhance cell survival.

[0189] Quantitative real-time PCR (qPCR) RNA is processed using the GenElute Mammalian Total RNA Miniprep Kit. A Miniprep Kit and On-Column DNAse I Digestion Set Extraction was performed using Sigma-Aldrich. cDNA synthesis was performed using Maxima First Strand cDN. A Synthesis Kit (Maxima First Strand cDNA Synthesis Kit) (Thermo Fisher Scientific) This was done using Applied Biosystems SYBR Green PCR Master Mix. Biosystems SYBR Green PCR Master Mix was used for qPCR. The sample was applied to Applied B The samples were run through an iosystems 7500 high-speed PCR machine. All samples were analyzed using technical duplication and housekey pins. The results were normalized for the gene porphobilinogen deaminase 1 (PBGD). The analysis was performed using the specified method. Please refer to Table 3 for primer sequences.

[0190] Flow cytometry For the analysis of EGFP-expressing cells, cells were incubated at 37°C for 5-10 minutes using TrypLE Select (Gibco). The cells were collected and a single-cell suspension was obtained. After washing with PBS, the cells were supplemented with DAPI (10 μg / ml). The cells were resuspended in ice-cold PBS and incubated on ice for 5 minutes. The cells were then subjected to cyanide ADP flow chromatography. Using a tometer, the level of EGFP expression in living cells (DAPI-negative) was determined. Staining and For the analysis of myosin heavy chain expression, cells were prepared using TrypLE Select (for EGFP expression analysis). (Regarding) Collect, wash once with PBS, fix, and dialysis with Cytofix / Cytoperm solution (BD Biosciences). Over-treated. Then the cells were washed and supplemented with 3% bovine serum albumin (BSA) Perm / wash. The cells were blocked overnight at +4°C with a cleansing buffer (BD Biosciences). PE-conjugated anti-MYH antibody (Table 4) was used. The staining was performed in Perm / wash buffer in the dark at +4°C for 1 hour. Three times with Perm / wash buffer. After washing, the cells were analyzed using a cyanide ADP flow cytometer to determine the level of MHC expression. Data analysis was performed using FlowJo (v10) and GraphPadPrism (v6).

[0191] Western blot Total cellular proteins were supplemented with a complete protease inhibitor (Roche) in CelLytic M (Sigma Extraction was performed using Aldrich, followed by the use of the Protein Quantification Kit-Rapid (Sigma-Aldrich). Quantitative analysis was performed by using NuPAGE LDS sample buffer and 4-12% N. The procedure was performed using uPAGE bis-tris precast gel (Invitrogen). Proteins were transferred onto PVDF. After transfer, the membrane was immersed in PBS supplemented with 0.05% Tween-20 (PBST) and 4% milk for 1 hour at room temperature. The membrane was blocked and incubated overnight with primary antibody in PBST 4% milk. Wash and incubate with HRP-conjugated secondary antibody (Sigma-Aldrich) in PBST 4% milk. erce ECL2 Western blotting substrate (Thermo Fisher Scientific) and incubation The X-ray Super RX film (Fujifilm) was then exposed to light.

[0192] immunocytochemistry The cells were fixed at room temperature for 20 minutes with 4% paraformaldehyde (diluted in PBS), and then, The cells were washed three times with PBS. Then, the cells were blocked with 10% donkey serum (Sigma-Aldrich). The cells were then permeabilized with 0.3% Triton X-100 (diluted with PBS) at room temperature for 20 minutes. Dilute appropriately in 2% donkey serum and 0.1% Triton X-100 (diluted in PBS) overnight at 4°C. The surface antigens PDGFRA, A2B5, and O4 were incubated with the primary antibody (supplementary experimental procedure). When staining, Triton X was removed at every step. After washing three times with PBS, 1% donkey blood was used. Corresponding robafluorophore-conjugated secondary antibodies (Alexa Fluor 488, 555, 488, 555) in PBS supplemented with the supernatant. Cells were incubated at room temperature for 1 hour in 568 and / or 647). The nuclei were treated with 4',6-diamidino Visualization was performed using -2-phenylindole (DAPI, Thermo Fisher Scientific) and EGFP. Expression and immunohistochemical staining were imaged using a Zeiss LSM 700 confocal microscope (Leica). βIII The percentage of tubulin-positive cells was measured using an inverted Olympus IX71 fluorescence microscope. Using fluorescence microscopy, at least 50 of the three biological replicates within three fields of view. By determining βIII tubulin expression in randomly selected DAPI-positive cells... It was calculated.

[0193] Statistical analysis was performed using GraphPad Prism (v6). The number of replicates and the statistical methods used were... The test results and examinations are described in the figure captions. Unless otherwise specified, the data is in flat format. It is presented as the mean ± SEM value.

[0194] Example 1: Dual targeting of EGFP To develop an inducible overexpression platform in hPSCs, the inventors used two Two components of the Tet-ON system were sequentially targeted to different GSHs. Third-generation constitutive expression. The rtTA of the human ROSA26 (hROSA26) gene locus was targeted using a CRISPR / Cas9n-based targeting strategy. The target was adjusted, and an inducible EGFP induction cassette was inserted into AAVS1 (Figure 1a; Figures 4a-c). hROSA26 and AA Targeting both VS1 molecules is highly efficient (Figures 4d-4f, Table 1), and contributes to hPSC genome stability and autologous function. It does not affect regeneration and differentiation (data not shown), and therefore does not affect rtTA-dependent cytotoxicity. They were not contradictory.

[0195] Next, the inventors possess one or two copies of each of the two induction cassettes. We selected a heavily GSH-targeted clone (Figure 5a). Homozygous targeting of rtTA was at a level approximately twice as high. This resulted in the rtTA protein (Figure 5b), and compared to heterozygous rtTA expression, after induction... This significantly increased the EGFP level (Figures 5c-5e). Furthermore, the homojugation of the inducible EGFP cassette was achieved. Clones with targeting showed higher and more uniform results compared to strains with heterozygous targeting. They showed EGFP levels (Figures 5c-5e). Importantly, all properly targeted strains were donkey It exhibits strong inducible EGFP expression, at least 20 times more pronounced compared to a strong constitutive CAG promoter. The performance was high (Figure 1b, Figures 5c-e). In summary, these results indicate that both systems of the Tet-ON system... The inventors' initial hypothesis was that targeting two copies of Rement would lead to maximum expression after induction. This supports the hypothesis. The peak in EGFP levels was reached approximately 4 days after induction, and doxycycline was removed. At times, expression was rapidly reversed (Figure 1c). Furthermore, EGFP expression was adjusted based on the doxycycline dose. Titration can be performed by stimulating the nucleotides (Figure 1d). Importantly, inducible EGFP expression is associated with hP Not only was it highly efficient in the SC stage, but it was also highly efficient during the differentiation into germ layers. Color photographic data is not shown; data is shown in Figures 6a-6d. Finally, the third-generation Tet-ON system. Consistent with known stringent transcriptional regulation, the same results were obtained by flow cytometry and qPCR, respectively. As defined, EGFP mRNA or protein can be detected in the absence of doxycycline. No background expression was observed (Figure 1b, Figure 6d). Overall, these results suggest that Tet- Dual GSH targeting of the ON system ensures optimal expression of the induction cassette in hPSCs and their derivatives. It has been established that this is a powerful strategy for that purpose.

[0196] Example 2: Induction of excitatory cortical neurons from hESCs and hiPSCs Previous studies have shown that these cells are related to the proneuron bHLH factor (ASCL1, N) in hPSCs. It can be easily induced by the overexpression of either GN2 or Neurod1 lentiviral. This was demonstrated. Therefore, the inventors fabricated OPTi-NGN2 hPSC (Figure 2a, Table 1). NGN2 Induction leads to rapid downregulation of pluripotency factors (Figure 7) and initiation of neuronal transcriptional programs (Figure 2). This resulted in (b). As early as three days after induction, the induced cells showed neurites (data not shown). ). After one week, all cells showed neuronal morphology and panneurotic cells such as βIII tubulin and MAP2. Marker proteins were expressed via marker proteins (Figure 2c). Quantitative RT-PCR revealed the presence of marker proteins such as BRN2 and FOXG1. Typical forebrain markers, as well as GRIA4, which indicates the identity of excitatory cortical neurons. The strong induction of glutamatergic neurons, including VGLUT2, was revealed (Figure 2b). In summary, these results suggest that, compared to conventional hPSC differentiation protocols, this approach leads to neuronal generation. Dramatic improvements in both speed and efficiency, as well as differentiation and lentivirus-based formulations. A substantial increase in efficiency and purity has been demonstrated with respect to both word programming protocols. Similar results were obtained with OPTi-NGN2 hiPSC, confirming the robustness of this method. Finally, the inventors demonstrated the efficiency of neural induction over an extended culture period of Opti-NGN2 hPSCs. No decrease was observed in this case (more than 25 passages, Figure 2c). Overall, the inventor Our results indicate that OPTi-NGN2 hPSC is capable of unlimited, highly scalable, rapid, and single-process neuronal development. It is a virus-free, almost definitive source for production and is used as an inexhaustible resource. It has been proven that this is possible.

[0197] Example 3: Production of skeletal muscle cells The transcription factor MYOD1, when overexpressed in various somatic cell types, induces myogenic differentiation. It is known that the ability of hPSCs to undergo MYOD1-induced myofibrillar forward programming is This is currently under discussion. The inventors have created OPTi-MYOD1 hPSCs (Table 1), but doxycycline It has been previously shown that induction of MYOD1 expression after rinsing promotes the conversion of hPSCs to skeletal muscle cells. We noticed that under the wide range of suggested culture conditions, we were able to induce almost complete cell death within 3-5 days. The cell reprogramming strategy involves combining extracellular signaling cues and the overexpression of transcription factors. It is widely established that improvements can be made by combining them, so the inventors This is a major signaling cascade involved in the formation of primitive streaks, somite formation, and myogenesis. By adjusting the [specific factor], a systematic screening of promyogenic factors was performed. (Inventor) They found that, in addition to MYOD1 overexpression, the addition of total trans retinoic acid (RA) resulted in 5 days after induction. By then, rapid and nearly complete ionization to myogenin and myosin heavy chain (MHC) double-positive muscle cells. We discovered that a conversion had occurred. The effect of RA was concentration-dependent, and the RA receptor isoform RAR The expression was mediated via α and RARβ and was consistent with the expression pattern of RA receptors during muscle development (Figure 8). This effect is thought to be independent of the mechanism of MYOD1 overexpression. The cells exhibit a typical spindle-shaped, elongated form, undergoing extensive cell fusion and composing mRNA and protein deposits. Strong myogenesis marker expression was observed at both levels (Figure 3b, Figures 9a-9c). When cetylcholine (ACh) or the selective ACh receptor agonist carbachol is added, complete This resulted in muscle fiber contraction and demonstrated the functionality of the induced muscle cells. Similar results were obtained with Opti-M Obtained with YOD1 hiPSC (data not shown). Importantly, the myogenic induction efficiency was extended. The amount did not decrease over the culture period (more than 50 subculturings, Figure 3d), therefore, the amount of this method Bust function and reproducibility were demonstrated. Finally, the inventors found that the level of the MYOD1 induction cassette was Positively correlated with conversion efficiency, the importance of robust gene delivery and lentiviral-mediated reprocessing I noticed that this method has advantages over the programming approach (Figure 10). Overall, the OPTi-MYOD1 forward programming strategy is the latest differentiation of hPSCs into skeletal muscle cells. It is approximately 7 times faster and 5 times more efficient than the protocol. (Previous forward programming...) Protocol (References by Tanaka, A. et al., PLoS One 8, e61540 (2013) and Abujarour, R. et al.) Compared to the study in Stem Cells Transl. Med. 3, 149-60 (2014), it is more efficient (95 Over 30-80%, does not contain randomly inserted induction cassettes, chemically defined, and complete. It is reproducible and more scalable.

[0198] These findings suggest that this method of controlling induced cassette expression in hPSCs is a high-sloop method. It can be used as an inexhaustible source for the large-scale production of sachets and homogeneous cell populations. This demonstrates that the induction speed and the purity of the desired target cells are currently unmatched by other methods. .

[0199] Example 4: Preparation of oligodendrocyte precursor and oligodendrocyte: Inducible SOX 1, either alone or in combination with OLIG2 in the form of a bicistronic expression cassette. OPTi-OX hPSCs possessing 0. Cells induced by SOX 10 alone showed oligodendrocytes 10 days after induction. These cells robustly expressed the drocyte precursor (OPC) marker O4, but these cells were Myelin The cells were unable to further differentiate into OLIG2-expressing cells and gradually died. In contrast, OLIG2- SOX10 dual-overexpressing cells transition from the O4-positive progenitor stage to the mature CNP / MBP-positive phenotype within 20 days of induction. It progressed easily. Furthermore, further analysis of marker protein expression revealed that mitogen PDGFaa and OPTi-OLIG2-SOX10 hPSC(Do) induced in oligodendrocyte medium supplemented with FGF2 The literature by uvaras et al. (2014) describes an OPC-like stage that is highly proliferative and co-expresses PDGFRA, A2B5, and O4. It was confirmed that they were the first to pass through the floor. These cells are highly proliferative and mitotic By culturing in the presence of fertilizer, it was possible to maintain the cells for at least three passages. Figure 12b). Therefore, the inventors identified these cells as i-OPCs and life cells compared to induced OPCs. It was named. Notably, after the removal of mitogens and the continued presence of doxycycline. Below, i-OPC is capable of myelin sheath formation (data not shown) of major myelin protein C They readily differentiate into mature oligodendrocytes expressing NP, PLP, MAG, MOG, and MBP in about one week. (Figures 12c-12d). In summary, these results indicate that the present invention relates to oligodendrocytes. Novel, robust, and rapid hPSC phosphating for the production of precursors and oligodendrocytes It has been demonstrated that this has enabled the development of code programming protocols.

[0200] Table 1: Summary of Genotype Determination Results [Table 1] (a) Incomplete targeting: No evidence of targeting (absence of bands in 5' and 3'-integrated PCR) There is evidence of targeting (the presence of a WT band in locus PCR) or 5'- or 3'-integration PCR. Incomplete size (b) Correct and targeted integration of plasmids through further random integration (3'-backbone (PCR band) (c) Correct and intended integration (HET, heterojunction; HOM, homojunction) (d) The percentage of clones that have the correct intended integration (with further integrations that are not intended) (not) (e) The percentage of clones that have the correct intended integration (with further unintended integrations) (or without) *The three numbers are from three different targeting experiments in hESC.

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

[0202] Table 3: List of primers for quantitative PCR [Table 3] TIFF2023134465000010.tif236170TIFF2023134465000011.tif42170

[0203] Table 4: List of antibodies [Table 4] TIFF2023134465000013.tif134170

[0204] Example 5: TET-ON Inducible Knockdown System Development of an inducible knockdown platform optimized for hPSCs The inventors have developed an hESC strain in which the EGFP transgene can be inductively silenced. The inventors created (Figure 14B). Therefore, the inventors proposed (1) CAG-tetR expression at the ROSA26 gene locus Set; and (2) CAG-EGFP transgene plus inducible EGFP shRNA cassette to the AAVS1 locus The tetracycline was targeted (Figure 14A, B). In the absence of tetracycline, higher levels of tetR were observed. When the protein is expressed, shRNA expression is more strongly suppressed. For this reason, the inventors, Multi-parameter RNA and codon optimization was performed on bacterial tetR cDNA, and the resulting codon-optimized te We used tR (OPTtetR) to create a new EGFP-inducible knockdown hESC strain (Figure 14B). This modification enabled a 10-fold increase in tetR expression compared to the standard sequence (STDt). etR; Figure 14D). Furthermore, homozygous expression of OPTtetR completely prevents shRNA leakage. While sufficient, it fully maintained efficient knockdown induction (Figure 14C). Inducible knockdown was rapid, reversible, and dose-responsive (Figure 14E, F). Finally, the inducible hESCs showed a normal karyotype (data not shown), and to create these strains It has been demonstrated that the necessary genome engineering does not alter genetic stability.

[0205] Based on these promising results, the inventors further proposed induction for POU5F1 / OCT4 or B2M. By creating hESCs that possess conductive shRNA, this method can be used in relation to endogenous genes. This was verified (data not shown). Notably, all the substrains analyzed (each gene) Six of the species showed robust inducible knockdown without significant shRNA leakage. Ikurin titration is used to identify the optimal concentration for partially or completely knocking down OCT4. As expected, the sharp decrease in OCT4 led to loss of pluripotency and neuroectoderm and endoderm development. This specifically induced Kerr's induction. Similar results were obtained in 20 additional OCT4-induced knockdown hE The results were obtained from the SC substrain, confirming the robustness and reproducibility of this method. Importantly, knock The creation of hESCs with strong and precisely controlled down is an antibiotic in mixed populations of cells. Immediately after selection, phenotypic analysis is performed to determine the individual colonies for clonal isolation. It was efficient enough to completely avoid the need for collection. Overall, this Based on these results, dual targeting of GSH with an optimized inducible knockdown system is h This app proves to be a powerful method for controlling gene expression in PSCs. Roach refers to optimized inducible knockdown as OPTiKD in this specification (Figure 14A).

[0206] Example 6 The ability to knock down genes in various differentiated cells is a successor to the previous inducible gene knockdown. This represents a significant advancement beyond a simple knockdown system. To thoroughly test this potential... The inventors found that in hPSCs differentiated into three germ layers, as well as in 13 fully differentiated cell types, Analysis of the effectiveness of the OPTiKD platform for knocking down the EGFP transgene in Nell. (Figure 15A). For both methods, qPCR analysis revealed that EGF was present in all strains tested. The potent and inducible knockdown of the P transcript was demonstrated (Figure 17). Microscopic observations were performed using EGFP. We confirmed a robust decrease in protein expression, and flow cytometry is most effective in most systems. The overall study showed a decrease of over 70% in EGFP fluorescence (data not shown).

[0207] Example 7 Development of an optimized inducible CRISPR / Cas9 knockout platform in hPSCs The inventors focused on developing an inducible knockout approach. The conductive CRISPR / Cas9 method allows for conditional overexpression of Cas9 in the presence of constitutively expressed gRNA. It depends. In this case, the control of Cas9 overexpression is tetracycline after doxycycline treatment. Phosphorus-regulated reverse trans-activator (rtTA) is involved in the Pol II-dependent tetracycline response. Rement (TRE) promoter (a fusion of multiple TET operons and a minimal CMV promoter) This is achieved by an activation method called TET-ON. This TET-ON platform is for specific humans While it has been successfully applied to various cell types, the inventors have found that this induction system targets AAVS1 GSH. Even after transformation, hPSCs are divided into multiple lineages (including cardiomyocytes, hepatocytes, and smooth muscle cells). Silencing was observed during the transformation (data not shown). The inventors developed an inducible shR Inducible gRNA cassettes and constitutive expression CAG promoters based on those developed for NA expression We explored the possibility of developing another improved method by combining it with advanced Cas9 (Figure). 18A, B). Therefore, the inventors have demonstrated that the fluorescent reporter gene can be knocked out in an inducible manner. We created an hESC strain that can be modified (Figure 18C). For this purpose, the inventors created a strain in which each transgene has two The AAVS1 locus, which is incorporated into one of the alleles, contains inducible EGFP gRNA and constitutive Cas9. We targeted the ROSA26-EGFPd2 reporter hESC, which possesses both. This dual-targeting approach It was rapid (less than 2 weeks) and efficient (over 90% of strains containing both transgenes). Notably, when individual clonal substrains were propagated in the presence of tetracycline... The inventors observed a decrease in EGFPd2 expression in all target cell lines, and in EGFPd2 homozygous cells. After tetracycline induction, at least one of the reporter genes can be detected as early as 5 days later. It showed a nearly homogeneous loss of P (as indicated by a 50% decrease in EGFPd2 fluorescence). Long-term treatment with lacyclin accelerates EGFPd2 fluorescence in up to 75% of EGFPd2 homozygous cells. This resulted in complete loss of sex (data not shown). Interestingly, the same AAVS1 locus... Co-expression of either two or three copies of the same EGFP gRNA cassette was observed in all analyzed clusters. To significantly improve the rate and efficiency of inducible EGFPd2 knockout in the Lone strain It took minutes. For example, simultaneous induction of three copies of the same gRNA after tetracycline treatment resulted in different results. This resulted in a 95% knockout efficiency. Importantly, inducible EGFPd2 knockout hESC Even if several gRNA copies are used, after long-term culture in the absence of tetracycline Furthermore, neither the proportion of EGFPd2-positive cells nor their fluorescence showed a significant decrease. It was demonstrated that the expression of conductive gRNA was strictly controlled. Finally, further gR against EGFPd2 NA studies revealed that the rate and efficiency of inducible knockout are strongly dependent on gRNA. In fact, the optimal sequence enabled up to 90% knockout just two days after induction. It was made possible. Notably, the most efficient gRNA also performed uncontrolled EGFPd2 knockout. However, this limitation leads to the induction of more stringent transcriptional control. This was avoided simply by adding a second TET operon to the conductive H1 promoter. In summary, this Our results indicate that the knockdown system supports inducible gRNA expression and broad gRNA efficacy. To enable well-controlled RISPR / Cas9 activity that surpasses expectations, it is easy to repurpose it. This demonstrates that it is possible to achieve this. To the best of the inventors' knowledge, this is the first based on inducible gRNA expression. This is a conditional CRISPR / Cas9 approach.

Claims

1. 1. A method for regulating the transcription of one or more transgenes encoding proteins in human cells ex vivo, comprising: a) inserting a transcriptional regulatory protein-encoding gene into a first gene safe harbor site; and b) inserting an inducible cassette into a second genetic safe harbor site, said inducible cassette comprising said one or more transgenes operably linked to an inducible promoter, said promoter being regulated by said transcriptional regulatory protein; Including, The method, wherein the first and second genetic safe harbor sites are different.

2. a transcriptional regulatory protein-encoding gene to be inserted into the first gene safe harbor site; and a human cell comprising a modified genome comprising an inducible cassette comprising one or more transgenes encoding one or more proteins operably linked to an inducible promoter inserted into a second genetic safe harbor site, The cell, wherein the inducible promoter is regulated by the transcriptional regulatory protein, and the first and second sites are different.

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

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

5. 5. The ex vivo method or cell of claim 4, wherein the transcriptional regulatory protein is operably linked to a constitutive promoter.

6. 6. The ex vivo method of any one of claims 1 or 3 to 5 or the cell of any one of claims 2 to 5, wherein the transcriptional regulatory protein is selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein comprising the DNA-binding domain of yeast GAL4 protein, a truncated ligand-binding domain of human progesterone receptor, and the activation domain of human NF-κB.

7. the activity of the rtTA is regulated by tetracycline or a derivative thereof, optionally doxycycline; and 7. The ex vivo method or cell of claim 6, wherein the inducible promoter comprises a Tet response element (TRE).

8. insertion into the first genetic safe harbor site and / or the second genetic safe harbor site, (i) has no deleterious effects on the inserted genetic material; or (ii) the ex vivo method of any one of claims 1 or 3 to 7 or the cell of any one of claims 2 to 7, which does not adversely affect said cell, such as by affecting the expression of any gene associated with cancer.

9. 9. The ex vivo method or cell of claim 8, wherein the first and second genomic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 (C-C motif chemokine receptor type 5) gene.

10. additional genetic material is inserted into the first / second genomic safe harbor site; Optionally, one or more of the following: a) suicide gene; b) selection marker; c) a reporter gene; and d) Non-coding RNA genes An ex vivo method according to any one of claims 1 or 3 to 9 or a cell according to any one of claims 2 to 9.

11. The ex vivo method of any one of claims 1 or 3 to 10 or the cell of any one of claims 2 to 10, wherein the cell is selected from a pluripotent stem cell, a somatic stem cell or a mature cell.

12. 12. The ex vivo method of any one of claims 1 or 3 to 11 or the cell of any one of claims 2 to 11, wherein said one or more transgenes encoding one or more proteins are suitable for forward programming of pluripotent stem cells into lineage-restricted, specific stem cells, progenitor cells or mature cells.

13. 13. The ex vivo method of any one of claims 1 or 3 to 12 or the cell of any one of claims 2 to 12, wherein said one or more proteins suitable for forward programming of pluripotent stem cells are master regulators or transcription factors.

14. the transcriptional regulatory protein-encoding gene is inserted into a first genetic safe harbor site on both chromosomes of the cell; and / or 14. The ex vivo method of any one of claims 1 or 3 to 13 or the cell of any one of claims 2 to 13, wherein the induction cassette is inserted into a second genetic safe harbor site on both chromosomes of the cell.

15. 15. The ex vivo method of any one of claims 1 or 3 to 14 or the cell of any one of claims 2 to 14, wherein a further induction cassette or transgene is inserted into a further GSH that is different from said first and second gene safe harbor (GSH).

16. A pharmaceutical composition comprising the cells of any one of claims 2 to 15 for use in therapy.

17. A pharmaceutical composition comprising the cells of any one of claims 2 to 15 for use in in vitro diagnostics.

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