New sites for secure genome integration and methods of use thereof

Abstract

The present disclosure relates to genetically modified cells that express one or more transgenes at a level of sustained expression from sites for safe genome integration and stable expression. The present disclosure also provides methods of making cells and nucleic acid vectors useful in making cells.

Description

[0001] Cross-references to related patent applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 336,248, filed on April 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. An electronic copy of the sequence listing, created on April 27, 2023, is named 025450_WO017_SL.xml and is 379,876 bytes in size. Background Art

[0005] Many efforts to safely integrate transgenes into the genome have been made at sites called "genomic safe harbors." Safe harbor sites in the genome are those sites where nucleic acids (e.g., foreign genes) can be introduced without interfering with the expression or regulation of adjacent genes, thereby not interfering with the normal function of the cell. Three genomic sites-AAVS1, CCR5, and ROSA26-are traditionally considered safe harbor sites and have been used for most targeted transgene integrations. AAVS1 is a region for rare genomic integration in the AAV genome, and it has been found to allow robust expression without disrupting cellular function. CCR5 was discovered by accident because the naturally occurring CCR5-delta-32 mutation results in an HIV-resistant phenotype; the gene's disposability makes it an ideal integration site. The ROSA26 locus was originally identified in mouse embryonic stem cells by a lentiviral gene capture method.

[0006] While these genomic safe harbor sites allow robust transgene expression in a given cell background, they may not support faithful transgene expression in other cell lines or after cell state changes. This is because the mutual interaction between the transgene and the genomic environment of the host cell can affect the expression of the transgene, resulting in attenuation or complete silencing of transgene expression (e.g., through DNA methylation). More critically, these genomic integration sites can also affect the expression of endogenous genes near the insertion site, thereby affecting normal host cell function. Summary of the invention

[0007] The present disclosure is based, at least in part, on the identification of intergenic sites in the genome that remain transcriptionally active in different cell types and different cell states (including stages of maturation), such that an exogenous nucleotide sequence of interest (e.g., a transgene encoding a protein or RNA) integrated therein remains expressed and functional as the cell undergoes proliferation and cell state changes.

[0008] Therefore, in one aspect, the present disclosure provides a genetically modified cell, such as a mammalian (such as a human) cell, comprising an exogenous nucleotide sequence integrated into a sustained transcriptionally active payload region (STAPLR) in the cell genome, wherein the STAPLR is selected from: the intergenic region between the RPL34 gene and the OSTC gene; the intergenic region between the ACTB gene and the FSCN1 gene; the intergenic region between the AKIRIN1 gene and the NDUFS5 gene; the intergenic region between the PRDX1 gene and the AKR1A1 gene; the intergenic region between the PTGES3 gene and the NACA gene; the intergenic region between the MLF2 gene and the PTMS gene; the intergenic region between the RAB13 gene and the RPS27 gene; the intergenic region between the JTB gene and the RAB13 gene. intergenic region; the intergenic region between the AKR1A1 gene and the NASP gene; the intergenic region between the NDUFS5 gene and the MACF1 gene; the intergenic region between the SRSF9 gene and the DYNLL1 gene; the intergenic region between the MYL6B gene and the MYL6 gene; the intergenic region between the GPX1 gene and the RHOA gene; the intergenic region between the HNRNPA2B1 gene and the CBX3 gene; the intergenic region between the ROMO gene and the RBM39 gene; the intergenic region between the PA2G4 gene and the RPL41 gene; and the intergenic region between the NDUFB10 gene and the RPS2 gene.

[0009] In some embodiments, the intergenic region between the RPL34 gene and the OSTC gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 1, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 1 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0010] In some embodiments, the intergenic region between the ACTB gene and the FSCN1 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 2, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 2 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0011] In some embodiments, the intergenic region between the AKIRIN1 gene and the NDUFS5 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 3, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 3 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0012] In some embodiments, the intergenic region between the PRDX1 gene and the AKR1A1 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 4, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 4 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0013] In some embodiments, the intergenic region between the PTGES3 gene and the NACA gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO:5, or a nucleotide sequence that is sufficiently similar to SEQ ID NO:5 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0014] In some embodiments, the intergenic region between the MLF2 gene and the PTMS gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 6, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 6 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0015] In some embodiments, the intergenic region between the RAB13 gene and the RPS27 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 7, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 7 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0016] In some embodiments, the intergenic region between the JTB gene and the RAB13 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 8, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 8 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0017] In some embodiments, the intergenic region between the AKR1A1 gene and the NASP gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 9, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 9 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0018] In some embodiments, the intergenic region between the NDUFS5 gene and the MACF1 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 10, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 10 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0019] In some embodiments, the intergenic region between the SRSF9 gene and the DYNLL1 gene contains a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 11, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 11 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0020] In some embodiments, the intergenic region between the MYL6B gene and the MYL6 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 12, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 12 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0021] In some embodiments, the intergenic region between the GPX1 gene and the RHOA gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 13, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 13 that the sequence function is intact (e.g., has no negative impact on the cell).

[0022] In some embodiments, the intergenic region between the HNRNPA2B1 gene and the CBX3 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 14, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 14 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0023] In some embodiments, the intergenic region between the ROMO gene and the RBM39 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 15, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 15 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0024] In some embodiments, the intergenic region between the PA2G4 gene and the RPL41 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 16, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 16 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0025] In some embodiments, the intergenic region between the NDUFB10 gene and the RPS2 gene comprises a nucleotide sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 97, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 97 to maintain the functional integrity of the sequence (e.g., having no negative impact on the cell).

[0026] Also provided herein are methods for producing these genetically modified mammalian cells, and DNA constructs for introducing a target nucleotide sequence into a new genomic integration site herein. Therefore, in one aspect, the disclosure provides a method for modifying mammalian cells, comprising integrating a target nucleotide sequence (i.e., an exogenous nucleotide sequence) into STAPLR as described herein. In some embodiments, the integration step is performed using the following techniques: CRISPR / Cas system; Cre / Lox system; FLP-FRT system; TALEN system; ZFN system; homing endonuclease; random integration; homologous recombination; transposase; or non-nuclease dependent viral vectors, optionally selected from retroviral vectors, adeno-associated virus (AAV) vectors, and lentiviral vectors. In a further embodiment, the CRISPR / Cas system comprises a guide RNA, and wherein STAPLR is (i) the intergenic region between the RPL34 gene and the OSTC gene, and the gRNA is selected from SEQ ID NOs: 25-32, (ii) the intergenic region between the ACTB gene and the FSCN1 gene, and the gRNA is selected from SEQ ID NOs: 33-54, (iii) the intergenic region between the AKIRIN1 gene and the NDUFS5 gene, and the gRNA is selected from SEQ ID NOs: 55-70, or (iv) the intergenic region between the PRDX1 gene and the AKR1A1 gene, and the gRNA is selected from SEQ ID NOs: 71-92.

[0027] In some embodiments, the CRISPR / Cas system comprises a gRNA-dependent nuclease of type I, type II, type III, type IV, or type V, or a variant thereof. In further embodiments, the CRISPR / Cas system comprises a gRNA-dependent nuclease selected from the group consisting of Cas9, Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas12, Cas13, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, C sn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx 14. Csx10, Csx16, CsaX, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, CasPhi, MAD7 and Csf4.

[0028] In another aspect, the present disclosure provides a DNA molecule comprising a nucleotide sequence of interest flanked by a 5' homology region (HR) and a 3' HR, wherein the 5' and 3' HR are at least 85% (e.g., at least 90, 95, 96, 97, 98 or 99%) homologous or 100% identical to the first genomic region (GR) and the second GR in the STAPLR described herein, respectively. In some embodiments, each 5' and 3' HR is independently about at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 base pairs in length. In some embodiments, each HR is 200 to 2000 (e.g., 300 to 2500, 400 to 2000, or 500 to 1500) base pairs long. In further embodiments, the 5' and 3' HRs are at least 90% (e.g., at least 95%) homologous to SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 93 and 94, or SEQ ID NOs: 95 and 96, respectively.

[0029] In some embodiments, the exogenous nucleotide sequence or the target nucleotide sequence comprises a transgene. In a further embodiment, the transgene comprises a coding sequence (e.g., for a protein or RNA) and one or more regulatory elements. In some embodiments, one or more regulatory elements include a constitutive or inducible promoter that directs transcription of the coding sequence. In some embodiments, the transgene encodes a therapeutic protein (e.g., a protein, which causes a disease such as a genetic disease due to its lack or defect; a cytokine; or a recombinant antigen receptor); a cell marker; or a protein that regulates the differentiation state or activity of a cell (e.g., a reprogramming factor). In some embodiments, the transgene encodes SOX10, IL-10, IL-12, CD19t or ThPOK.

[0030] In some embodiments of the present disclosure, the mammalian cell is a human cell. In some embodiments, the mammalian cell (eg, human cell) is a pluripotent stem cell (PSC; eg, induced PSC (iPSC) or embryonic stem cell (ESC)). In some embodiments, the mammalian cell (e.g., human cell) is a) a cell in the immune system (e.g., a T cell, a natural killer cell, a dendritic cell, a macrophage / monocyte, or a hematopoietic progenitor or precursor cell thereof); b) a cell in the cardiovascular system (e.g., a ventricular cardiomyocyte, a node cell, or a cardiac progenitor or precursor cell thereof); c) a cell in the metabolic system (e.g., a hepatocyte or a pancreatic β cell, or a progenitor or precursor cell thereof); d) a cell in the central nervous system (e.g., a sensory neuron, a motor neuron, an interneuron, a microglia, an oligodendrocyte, or a progenitor or precursor cell thereof); e) a muscle cell (e.g., a skeletal muscle cell or a smooth muscle cell, or a progenitor or precursor cell thereof); f) an adipocyte, or a progenitor or precursor cell thereof); or g) a cell in the ocular system (e.g., a retinal pigment epithelial cell, a photoreceptor, or a progenitor or precursor cell thereof). Additional cell types of the present disclosure include those described below.

[0031] Also provided herein is a pharmaceutical composition comprising the genetically engineered cells and a pharmaceutically acceptable vector described herein, and a gene editing system comprising the DNA molecules disclosed herein and the necessary gene editing system for integrating the target nucleotide sequence (e.g., nuclease and gRNA) on the DNA molecule into STAPLR.

[0032] On the other hand, the present disclosure provides a method for identifying a persistent transcriptionally active payload region (STAPLR) in a mammalian cell genome, the method comprising: (i) performing single-cell RNA sequencing analysis on a set of two or more mammalian cell types, wherein the sequencing analysis assigns a unique transcriptome to each cell type; (ii) assigning a prevalence score (Prevalence Score) to a constitutive gene in the transcriptome, wherein the prevalence score represents the fraction of mammalian cell types that contain at least one transcript of the gene in the set of mammalian cell types; (iii) identifying neighboring genes of the constitutive gene in the genome of the mammalian cell, wherein the neighboring gene does not overlap with the constitutive gene; (iv) determining a neighbor score (Neighbor Score) for a non-overlapping gene pair or a region containing three or more genes identified in step (iii), wherein the neighbor score is the product of the prevalence scores of the individual genes in a pair or a region; (v) ranking the neighbor scores; and (vi) selecting a pair of non-overlapping genes or a region containing three or more non-overlapping genes based on a high ranking, thereby identifying the intergenic region between the selected gene pair or region as a STAPLR. In some embodiments, the method further includes (vii) selecting a targetable intergenic subregion in STAPLR; and (viii) inserting a transgene in the selected subregion, wherein the transcription of the transgenic or gene circuit is continuous. In some embodiments, the targetable subregion includes: no known promoter or enhancer region, a minimum number of conserved regions, repeat regions, epigenetic markers and / or enzymatic hypersensitive regions, and / or the nuclease is a CRISPR nuclease. In some embodiments, the length of the intergenic region is at least 30 (e.g., at least 40, at least 50, at least 75, or at least 100) base pairs, and / or does not include or includes a minimum number of promoter regions, CpG islands, H3K4Me1 epigenetic markers, H3K4Me3 epigenetic markers, H3K27Ac epigenetic markers, DNase I hypersensitive regions, conserved regions, or repeat regions.

[0033] Other features, purposes and advantages of the present invention are apparent in the detailed description below. However, it should be understood that the detailed description, although indicating embodiments and aspects of the present invention, is given only by way of illustration, not limitation. Based on the detailed description, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1To show the insertion / deletion editing percentage obtained after Sanger sequencing test using Synthego's ICE Analysis Tool. For each different STAPLR site, three different gRNAs were tested, and the gRNA with the highest insertion / deletion editing percentage was circled. The horizontal solid line represents the average insertion / deletion editing percentage of three different gRNAs for each STAPLR site.

[0035] Figure 2 Figure 2 shows the sequence encoding the 2A peptide and the sequence encoding the Tet-On 3G version of rtTA integrated into the GAPDH locus. The left and right homology arms are designed to enable the transgene to be integrated in frame to the 5' of the stop codon of GAPDH immediately adjacent to (immediately). This allows the expression of rtTA to be under the control of the endogenous GAPDH promoter. iPSCs edited with the targeting construct constitutively express the rtTA protein.

[0036] Figure 3 Figure 2 shows the integration of each of the four STAPLR targeting constructs containing the pTRE3G-eGFP-Sv40 transgene flanked by left and right homology arms into each STAPLR site in iPSCs constitutively expressing the rtTA protein. The addition of doxycycline allows the binding of the rtTA protein and the activation of GFP expression from the TRE3G promoter.

[0037] Figure 4 It is a group of fluorescence microscopy images, which describes the expression of GFP in the cell colonies merged with doxycycline for 24 hours. 24 hours after iPSCs were nuclear transfected with STAPLR targeting construct and corresponding RNP, doxycycline was added to the culture medium. GFP was not observed in cells that did not receive doxycycline. The control iPSCs that were constitutively expressed rtTA and were not nuclear transfected with STAPLR targeting construct and RNP did not express GFP either.

[0038] Figure 5 It is a group of fluorescence microscopy images, which describes the expression of GFP in the cell colonies that have been merged to receive doxycycline for 6 days. 24 hours after iPSCs were nuclear transfected with STAPLR targeting construct and corresponding RNP, doxycycline was added to the culture medium. GFP was not observed in cells that did not receive doxycycline. The control iPSCs that were constitutively expressed rtTA and were not nuclear transfected with STAPLR targeting construct and RNP did not express GFP either.

[0039] Figure 6Figure 2 is a set of flow cytometry histograms depicting the degree of induction of GFP expression over time in four different clonally derived STAPLR iPSC lines at different concentrations of doxycycline. Cells were collected for analysis at 0, 3, 8, 24, 48, and 68 hours after doxycycline administration.

[0040] Figure 7 A set of flow cytometry histograms depicting the degree of induction of GFP expression over time in four different clonally derived STAPLR iPSC lines after treatment with 2 μg / ml doxycycline. The left panel shows PRDX1-AKR1A1, ACTB-FSCN1, and RPL34-OSTC STAPLR lines and a wild-type, unedited iPSC control line that were not treated with doxycycline or were treated with doxycycline for 72 hours. The right panel shows the AKIRIN1-NDUFS5 STAPLR line that was not treated with doxycycline or was treated with doxycycline for 6 days.

[0041] Figure 8 It is a set of flow cytometry histograms, which describes the degree of induction of GFP expression in four different clone-derived STAPLR iPSC lines differentiated into myeloid progenitor cells after treatment with 2 μg / ml doxycycline. Doxycycline was added to the culture medium on the 12th day of differentiation. The left figure shows the PRDX1-AKR1A1, ACTB-FSCN1 and RPL34-OSTC STAPLR lines and wild-type unedited iPSC control lines that were not treated with doxycycline or treated with doxycycline for 72 hours after 15 days of myeloid differentiation. The right group shows the AKIRIN1-NDUFS5 STAPLR line that was not treated with doxycycline or treated with doxycycline for 6 days after 18 days of myeloid differentiation.

[0042] Fig. 9 is a set of flow cytometry dot plots showing the expression of myeloid progenitor markers CD45, CD14 and CX3CR1 in the non-adherent bone marrow population of the STAPLR-targeted iPSC line that has been differentiated for more than 30 days. Cells of the CD14 and CX3CR1 groups were gated on CD45-positive cells.

[0043] Fig.10 Figure 2 is a set of flow cytometry histograms depicting the extent of induction of GFP expression in non-adherent myeloid progenitors in four differentiated clonally derived STAPLR iPSC lines and a wild-type unedited iPSC control line following treatment with 2 μg / ml doxycycline. Doxycycline was added to the culture medium for 6 days after 30 days of differentiation.

[0044] Fig.11Diagram showing integration of a targeting construct containing the pTRE3G-CD19t-IL12 transgene flanked by left and right homology arms to allow integration at the PRDX1-AKR1A1STAPLR locus. This construct was transfected into iPSCs constitutively expressing rtTA protein from the GAPDH endogenous promoter.

[0045] Fig.12 A set of photographs showing live cell imaging of CD19t (truncated to prevent intracellular signaling) staining in pooled cell samples after targeting with the PRDX1-AKR1A1 pTRE3G-CD19t-IL12 donor template or in clonal cell populations after single cell clonal density seeding after treatment with 2 μg / mL doxycycline for 48 hours compared to untreated cells. Panel A shows cells after targeting with Cpf1-based RNPs. Panel B shows cells after targeting with Cas9-based RNPs.

[0046] Fig.13 is a set of fluorescence microscopy images depicting the expression of GFP in a pooled population of cells that had received doxycycline for 24 hours. Doxycycline was added to the culture medium 48 hours after iPSCs were nucleofected with a targeting construct for PRDX1-AKR1A1 site 2 and three different RNPs containing three different gRNAs targeting site 2. GFP was not observed in cells that did not receive doxycycline.

[0047] Fig.14 is a set of fluorescence microscopy images depicting the expression of GFP in a pooled population of cells that had received doxycycline for 24 hours. Doxycycline was added to the culture medium 24 hours after iPSCs were nucleofected with a PRDX1-AKR1A1 site 3 targeting construct and three different RNPs containing three different gRNAs targeting site 3. No GFP was observed in cells that did not receive doxycycline.

[0048] Fig.15 Figure 1 is a set of flow cytometry histograms depicting the degree of induction of GFP expression in a pooled population of cells treated with 2 μg / ml doxycycline. Doxycycline was added to the culture medium 48 hours after iPSCs were nucleofected with a targeting construct for site 2 of PRDX1-AKR1A1 and three different RNPs containing three different gRNAs targeting site 2. Flow cytometric analysis was performed 5 days after doxycycline treatment. GFP was not observed in cells that did not receive doxycycline and in parental GAPDH::rtTA iPSCs that did not receive the targeting construct and RNP.

[0049] Fig.16Figure 1 is a set of flow cytometry histograms depicting the degree of induction of GFP expression in a pooled population of cells treated with 2 μg / ml doxycycline. Doxycycline was added to the culture medium 24 hours after iPSCs were nucleofected with a targeting construct for site 3 of PRDX1-AKR1A1 and three different RNPs containing three different gRNAs targeting site 3. Flow cytometric analysis was performed 6 days after doxycycline treatment. GFP was not observed in cells that did not receive doxycycline and in parental GAPDH::rtTA iPSCs that did not receive the targeting construct and RNP. DETAILED DESCRIPTION

[0050] Genetically engineered cells are important tools for cell therapy. However, as time goes by, the artificial gene circuits in engineered cells are often destroyed by transgenic silencing because the cells undergo proliferation, or the cell state or the in vivo environment changes. Therefore, it is necessary to identify the genomic regions that are safe for transgenic integration, and also provide a chromatin map that is open to transcription across cell types, cell states, and in vivo environments. Transgenic integration into such a site will allow the transgenic to maintain transcriptional activity during the life of the cell therapy product.

[0051] Provided herein are compositions (e.g., nucleic acid molecules and cells) and methods for genomic (genetic) engineering cells to achieve expression of transgenes across various cells or differentiation states without affecting endogenous gene expression that may be harmful to the cells or therapeutic purposes of the cells in cell therapy. Provided compositions and methods are based, at least in part, on the identification of chromatin profiles that contain persistent transcriptionally active payload regions (STASTAPLRs) that remain transcriptionally active across cell types and differentiated cell states.

[0052] I.STAPLR

[0053] The inventors have found that, regardless of cell type and / or even when cell state undergoes changes (e.g., differentiation state, maturity or active state), certain intergenic regions in mammalian genomes allow transgenics integrated therein to have consistent expression levels. This discovery greatly expands the scope of genomic sites, wherein transgenics can be stably integrated and their expression can be maintained under the cell state of change. Therefore, this discovery solves the long-term problem in transgenic expression, such as in the context of cell therapy. These intergenic regions are referred to herein as "continuous transcriptional active payload region (STAPLR)", wherein "payload (payload)" or "genomic payload (genomic payload)" refers to one or more exogenous or heterologous nucleotide sequences introduced into this region. STAPLR includes an open chromatin map for landing on the genome payload. The chromosomal DNA in STAPLR is in a conformation accessible to gene editing system elements, and allows the integration of genetic material. In some cases, STAPLR is in the vicinity of transcriptionally active genes.

[0054] An application of this discovery is to effectively produce cells (e.g., therapeutic cells), which are first genetically modified, and then such as by differentiation or dedifferentiation to change the cell state. For example, the genetic engineering method of the present invention can be applied to iPSC, which is then differentiated into various cell types. In the past, when iPSC was engineered to introduce transgenes into its genome, and then differentiated into the desired cell type, transgenes could become inactive when iPSC differentiated. However, the transgenes integrated into STAPLR disclosed herein will not become inactive after iPSC differentiation. Therefore, STAPLR provides a universal "landing pad (landing pad)" for transgenic expression.

[0055] Such stability in transgene expression is also advantageous after administration of therapeutic cells in cell therapy to a subject in need thereof (eg, a human patient), which may encounter different and varying circumstances that shut down a transgene integrated elsewhere.

[0056] In addition, transgenic integration is in the intergenic region, rather than in the gene, the expression of adjacent genes or regulation will be caused to minimum destruction, and therefore allow the normal function of genetically engineered cells. The transgenic integration at the STAPLR place has also reduced the risk of causing undesirable influence (as, activating oncogenes or destroying essential genes, for example tumor suppressor genes) in cells. In addition, STAPLR with continuous transcriptional activity state allows testing and use of a wider range of regulatory elements (as, promoter and enhancer).

[0057] As used herein, an "intergenic region" is a nucleotide sequence located between two adjacent genes. An intergenic region can be of various sizes. For example, the length of the intergenic region can be at least 30, 40, 50, 75, or 100 base pairs. In some embodiments, the length of the intergenic region can be at least 150, 200, 300, 400, 500, 750, or 1000 base pairs. In some embodiments, the length of the intergenic region can be at least 1500, 2000, 2500, 3000, 3500, 5000, or 10000 base pairs. In some embodiments, the length of the intergenic region can be at least 15000, 20000, 30000, 40000, 50000, 75000, or 100000 base pairs. In some embodiments, the intergenic region is 30 base pairs to 100,000 base pairs in length. In some embodiments, the intergenic region is 50 base pairs to 75,000 base pairs in length. In some embodiments, the intergenic region is 75 base pairs to 70,000 base pairs in length.

[0058] STAPLR of the present disclosure includes, but is not limited to (NCBI Gene IDs of human genes shown in brackets): the intergenic region between the RPL34 gene (Gene ID: 6164) and the OSTC gene (Gene ID: 58505), the intergenic region between the ACTB gene (Gene ID: 60) and the FSCN1 gene (Gene ID: 6624), the intergenic region between the AKIRIN1 gene (Gene ID: 79647) and the NDUFS5 gene (Gene ID: 4725), the intergenic region between the PRDX1 gene (Gene ID: 5052) and the AKR1A1 gene (Gene ID: 10327), the intergenic region between the PTGES3 gene (Gene ID: 10728) and the NACA gene (Gene ID: 4666), the intergenic region between the MLF2 gene (Gene ID: 8079) and the PTMS gene (Gene ID: 5763), the intergenic region between the RAB13 gene (Gene ID: 5872) and the RPS27 gene (Gene ID: 5874). The intergenic region of the 3 genes was 4725 (Gene ID: 4725) and MACF1 (Gene ID: 23499), the intergenic region between SRSF9 gene (Gene ID: 8683) and DYNLL1 gene (Gene ID: 8655), the intergenic region between MYL6B gene (Gene ID: 140465) and MYL6 gene (Gene ID: 4637), the intergenic region between GPX1 gene (Gene ID: 2876) and RHOA gene (Gene ID: 387), the intergenic region between HNRNPA2B1 gene (Gene ID: 3181) and CBX3 gene (Gene ID: 3182). In some embodiments, the gene herein refers to a human gene, and the mammalian cell is a human cell.

[0059] The start and end genomic coordinates and sizes of the above-mentioned STAPLR intergenic region in the human genome are listed below in Table 1. The coordinates are defined by information available in the RefSeq database of NCBI.

[0060] Table 1. Intergenic regions between selected genes

[0061]

[0062]

[0063] Due to differences between humans and between mammalian species, the intergenic regions between the above gene pairs may differ to some extent from the corresponding SEQ ID NOs shown in Table 1.

[0064] In some embodiments, the intergenic region between the RPL34 gene and the OSTC gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, or a nucleotide sequence sufficiently similar to SEQ ID NO: 1 such that the intergenic region retains the function of SEQ ID NO: 1, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the PL34 gene and the OSTC gene remains intact (e.g., has no negative impact on the cell).

[0065] In some embodiments, the intergenic region between the ACTB gene and the FSCN1 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2, or a nucleotide sequence sufficiently similar to SEQ ID NO: 2 such that the intergenic region retains the function of SEQ ID NO: 2, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the ACTB gene and the FSCN1 gene remains intact (e.g., has no negative impact on the cell).

[0066] In some embodiments, the intergenic region between the AKIRIN1 gene and the NDUFS5 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 3 such that the intergenic region retains the function of SEQ ID NO: 3, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the AKIRIN1 gene and the NDUFS5 gene remains intact (e.g., has no negative impact on the cell).

[0067] In some embodiments, the intergenic region between the PRDX1 gene and the AKR1A1 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4, or a nucleotide sequence sufficiently similar to SEQ ID NO: 4 such that the intergenic region retains the function of SEQ ID NO: 4, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the PRDX1 gene and the AKR1A1 gene remains intact (e.g., has no negative impact on cells).

[0068] In some embodiments, the intergenic region between the PTGES3 gene and the NACA gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:5, or a nucleotide sequence sufficiently similar to SEQ ID NO:5 such that the intergenic region retains the function of SEQ ID NO:5, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the PTGES3 gene and the NACA gene remains intact (e.g., has no negative impact on cells).

[0069] In some embodiments, the intergenic region between the MLF2 gene and the PTMS gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 6 such that the intergenic region retains the function of SEQ ID NO: 6, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the MLF2 gene and the PTMS gene remains intact (e.g., has no negative impact on cells).

[0070] In some embodiments, the intergenic region between the RAB13 gene and the RPS27 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 7, or a nucleotide sequence sufficiently similar to SEQ ID NO: 7 such that the intergenic region retains the function of SEQ ID NO: 7, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the RAB13 gene and the RPS27 gene remains intact (e.g., has no negative impact on the cell).

[0071] In some embodiments, the intergenic region between the JTB gene and the RAB13 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 8, or a nucleotide sequence sufficiently similar to SEQ ID NO: 8 such that the intergenic region retains the function of SEQ ID NO: 8, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the JTB gene and the RAB13 gene remains intact (e.g., has no negative impact on the cell).

[0072] In some embodiments, the intergenic region between the AKR1A1 gene and the NASP gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9, or a nucleotide sequence sufficiently similar to SEQ ID NO: 9 such that the intergenic region retains the function of SEQ ID NO: 9, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the AKR1A1 gene and the NASP gene remains intact (e.g., has no negative impact on the cell).

[0073] In some embodiments, the intergenic region between the NDUFS5 gene and the MACF1 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 10, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 10 such that the intergenic region retains the function of SEQ ID NO: 10, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the NDUFS5 gene and the MACF1 gene remains intact (e.g., has no negative impact on cells).

[0074] In some embodiments, the intergenic region between the SRSF9 gene and the DYNLL1 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 11, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 11, such that the intergenic region retains the function of SEQ ID NO: 11, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the SRSF9 gene and the DYNLL1 gene remains intact (e.g., has no negative impact on cells).

[0075] In some embodiments, the intergenic region between the MYL6B gene and the MYL6 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 12, or a nucleotide sequence that is sufficiently similar to SEQ ID NO: 12 such that the intergenic region retains the function of SEQ ID NO: 12, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the MYL6B gene and the MYL6 gene remains intact (e.g., has no negative impact on the cell).

[0076] In some embodiments, the intergenic region between the GPX1 gene and the RHOA gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 13, or a nucleotide sequence sufficiently similar to SEQ ID NO: 13 such that the intergenic region retains the function of SEQ ID NO: 13, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the GPX1 gene and the RHOA gene remains intact (e.g., has no negative impact on the cell).

[0077] In some embodiments, the intergenic region between the HNRNPA2B1 gene and the CBX3 gene comprises a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 14, or a nucleotide sequence sufficiently similar to SEQ ID NO: 14, such that the intergenic region retains the function of SEQ ID NO: 14, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the HNRNPA2B1 gene and the CBX3 gene remains intact (e.g., has no negative impact on the cell).

[0078] In some embodiments, the intergenic region between the ROMO gene and the RBM39 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15, or a nucleotide sequence sufficiently similar to SEQ ID NO: 15 such that the intergenic region retains the function of SEQ ID NO: 15, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the ROMO gene and the RBM39 gene remains intact (e.g., has no negative impact on the cell).

[0079] In some embodiments, the intergenic region between the PA2G4 gene and the RPL41 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16, or a nucleotide sequence sufficiently similar to SEQ ID NO: 16 such that the intergenic region retains the function of SEQ ID NO: 16, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the PA2G4 gene and the RPL41 gene remains intact (e.g., has no negative impact on cells).

[0080] In some embodiments, the intergenic region between the NDUFB10 gene and the RPS2 gene comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 16, or a nucleotide sequence sufficiently similar to SEQ ID NO: 16 such that the intergenic region retains the function of SEQ ID NO: 97, i.e., the function (e.g., transcriptional regulation) of the intergenic region between the NDUFB10 gene and the RPS2 gene remains intact (e.g., has no negative impact on the cell).

[0081] The percent identity of two nucleotide sequences can be determined by using the default parameters In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, or 90%) of the reference sequence.

[0082] II. Integration of foreign sequences into STAPLR

[0083] A. Integration site

[0084] The exogenous nucleotide sequence of interest can be integrated at any site within the STAPLR. For example, the integration site or the junction between the exogenous sequence and the adjacent endogenous sequence can be located in the first half or the second half of the STAPLR; in the 5', middle or 3' third of the STAPLR; or in the first, second, third or fourth quarter of the STAPLR. In some embodiments, the integration site of the exogenous sequence or the junction between the exogenous sequence and the adjacent endogenous sequence is located within the STAPLR and is at least 10, 20, 30, 40, 50, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 10000, 15000 or 20000 base pairs away from the nearest gene, i.e., from the 5' or 3' border of the STAPLR (e.g., from the start or end coordinates shown in Table 1).

[0085] In a single genome, one or more exogenous nucleotide sequences can be integrated into one or more STAPLRs. In some embodiments, one or more (e.g., two, three or four) exogenous nucleotide sequences can be integrated into one or more sites within a single given STAPLR. In some embodiments, more than one STAPLR in a targeted single genome is used to integrate exogenous nucleotide sequences.

[0086] In some embodiments, as described in WO 2021 / 072329, exogenous sequences are introduced into at least one STAPLR and at least one persistent transgenic expression locus (STEL). The STEL site is a locus of an endogenous gene that is robustly and consistently expressed in a pluripotent state and during differentiation (e.g., as tested by single-cell RNA sequencing (scRNAseq) analysis). Although STAPLR can be associated with the STEL site, it does not need to be associated with the STEL site. The STEL site can be identified from single-cell RNA sequence data. The defining feature of the desired STEL site is the ubiquity of expression. STEL sites can be identified by analyzing the expression of candidate loci in different cell types and cell maturation states, such as PSCs and PSC-derived dopamine neurons (and progenitor cell selection states), microglia (and progenitor cell selection states), and cardiomyocytes (and cardiomyocyte progenitor cell selection states). Such STEL analysis can be improved by adding publicly available single-cell RNA sequencing data of adult human tissues. STELs include, but are not limited to, some housekeeping genes that are active in multiple cell types, such as those involved in gene expression (e.g., transcription factors and histones), cellular metabolism (e.g., GAPDH and NADH dehydrogenase), or cellular structure (e.g., actin), or those encoding ribosomal proteins (e.g., large or small ribosomal subunits, such as RPL13A, RPLP0, and RPL7).Examples of STELs are genes encoding ribosomal proteins, such as RPL genes (e.g., RPL13A, RPLP0, RPL10, RPL13, RPS18, RPL3, RPLP1, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22) and RPS genes (e.g., RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPS11); genes encoding mitochondrial proteins (e.g., MT-CO1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2); genes encoding actin (ACTG1 and ACTB); genes encoding eukaryotic translation factors (e.g., EEF1A1, EEF2, and EIF1); and genes encoding histones (e.g., H3F3A and H3F3B). Additional STELs are those encoding proteins involved in focal adhesion, cell-substrate adhesion junctions, cell-substrate junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, or anchorage junctions. Additional examples of STELs are FTL, FTH1, TPT1, TMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0087] In some embodiments, in a single mammal (e.g., human) genome, an exogenous sequence is introduced into STAPLR, such as RPL34-OSTC or PRDX1-AKR1A1 STAPLR and STEL, such as the GAPDH locus. In some embodiments, an exogenous sequence is introduced into multiple STAPLRs in a single genome, such as RPL34-OSTC and PRDX1-AKR1A1 STAPLR.

[0088] The integration site of the exogenous nucleotide sequence may be within STAPLR or in a gene sequence adjacent to STAPLR (e.g., in an exon, intron, or UTR of a gene). In some embodiments, the endonuclease produces DNA breaks within STAPLR. In other embodiments, the endonuclease produces DNA breaks in a gene adjacent to STAPLR, so that after integration, the exogenous nucleotide sequence is still integrated within STAPLR. In some embodiments, screening for inappropriate integration events may be performed according to the method described in WO 2021 / 226151, wherein DNA breaks are introduced into exons of genes adjacent to STAPLR and essential for cell survival, and those cells in which integration is not properly achieved cannot survive.

[0089] B. Integration Methods

[0090] Any genome integration method can be used to utilize STAPLR as described herein.In some embodiments, the integration of exogenous nucleotide sequence in STAPLR is realized by using genome editing system, and the genome editing system is selected from CRISPR / Cas system, Cre / Lox system, FLP-FRT system, transcription activator sample effector nuclease (TALEN) system, zinc finger nuclease (ZFN) system, homing endonuclease, sequence-specific endonuclease, random integration (such as, by transposon), meganuclease, homologous recombination, transposase and non-nuclease dependent viral vector (such as, retrovirus, AAV or slow virus vector).In some embodiments, integration will not cause the disappearance of endogenous sequence in this region, and / or the addition of nucleotide sequence except exogenous donor sequence to be integrated.In some embodiments, integration will cause (non-donor sequence) insertion and / or disappearance (indel) at integration site.

[0091] In some embodiments, the exogenous sequence can be incorporated into the STAPLR site by homologous recombination at the DNA break generated by a suitable nuclease (such as a CRISPR-associated nuclease), for example, the CRISPR-associated nuclease can be a Cas nuclease, and the Cas nuclease is selected from, but not limited to: type I (e.g., subtype IA, IB, IC, IC variant, ID, IE, IF, IF variant 1 or IF variant 2), type II (e.g., subtype II-A, II-B or II-C), type III (e.g., subtype III-A, III-B or III-B variant), type IV or type V Cas protein or its variant. In some embodiments, the nuclease is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12 (e.g., Cas12a or Cpf1 or Cas12b), Cas13, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, CasPhi, MAD7, Csf4, and homologs thereof or modified forms thereof (e.g., truncated forms or variants of wild-type Cas proteins having nuclease activity).

[0092] In some embodiments, the Cas endonuclease is a Cpf1 (Cas12a) endonuclease, or a variant, derivative or fragment thereof, such as Cpf1 derived from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1, including improved variants, such as enAsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpf1), Lachnospiraceae bacterium MC2017 (Lb3Cpf1), Moraxella oxeye bovoculi 237 (MbCpf1), or Prevotella disiens (PdCpf1).

[0093] In some embodiments, the Cas endonuclease is a Cas9 protein or a variant, derivative or fragment thereof. In some embodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9 or D10A nickase.

[0094] In some embodiments, the Cas endonuclease is a V-type RNA programmable nuclease, as disclosed in WO 2022 / 258753.

[0095] In some embodiments, the Cas endonuclease is a MAD nuclease, such as a MAD7 nuclease, as disclosed in U.S. Pat. No. 10,337,028.

[0096] Non-limiting examples of suitable endonucleases are listed in Table A below.

[0097] Table A Exemplary endonucleases

[0098]

[0099]

[0100]

[0101]

[0102] In some embodiments, the CRISPR / Cas system comprises a gRNA-dependent nuclease (or its coding sequence) that targets a selected intergenic region, a gRNA (or its coding sequence), and a donor DNA comprising an exogenous nucleotide sequence.

[0103] In some embodiments, STAPLR is the intergenic region between the RPL34 gene and the OSTC gene, and the gRNA is selected from SEQ ID NOs: 25-32.

[0104] In some embodiments, STAPLR is the intergenic region between the ACTB gene and the FSCN1 gene, and the gRNA is selected from SEQ ID NOs: 33-54.

[0105] In some embodiments, STAPLR is the intergenic region between the AKIRIN1 gene and the NDUFS5 gene, and the gRNA is selected from SEQ ID NOs: 55-70.

[0106] In some embodiments, STAPLR is a genetic intergenic region between the PRDX1 gene and the AKR1A1 gene, and the gRNA is selected from SEQ ID NOs: 71-92.

[0107] C. Foreign nucleotide sequences

[0108] In some embodiments, the exogenous nucleotide sequence of interest for integration may comprise a transgene or RNA encoding a protein (including a peptide as used herein). The transgene may comprise a coding sequence for a gene product, and optionally one or more transcriptional regulatory elements. In some embodiments, the transgene comprises one or more regulatory elements, wherein the one or more regulatory elements may optionally be operably linked to the coding sequence.

[0109] Non-limiting examples of regulatory elements are promoters, enhancers, silencers, chromatin insulators, intronic sequences, Kozak sequences, ubiquitous chromatin opening elements (UCOEs), transcription activator binding elements, sequences that enhance gene expression or RNA stability (e.g., WPRE elements), polyadenylation signal sequences (e.g., SV40 polyA signals), and the like.

[0110] In some embodiments, the promoter directing transgene expression is a constitutive promoter, including but not limited to EF1a, EFS, UBC, PGK, CAGGS, CMV, SV40, B2M and ROSA26 promoters. In some embodiments, the promoter is a cell type-specific, tissue-specific or cell lineage-specific promoter. For example, the promoter may be a tyrosine hydroxylase promoter for dopaminergic neurons; an Hb9 promoter for motor neurons; a SIRPA promoter for cardiomyocytes; a CD14, CD33, CD45 or CD11b promoter for myeloid lineage cells; or a CD3, FOXP3, CD25, CD8 or CD4 promoter for T lymphocytes. In some embodiments, expression of the transgene is under the control of an inducible promoter (e.g., a lac operon, which can be triggered by isopropyl β-D-1-thiogalactopyranoside (IPTG); a TRE promoter, which can be triggered by tetracycline and its derivatives).

[0111] In some embodiments, the exogenous sequence comprises one or more regulatory elements that respond to factors expressed from another site (e.g., from an endogenous gene or transgene integrated at STEL or STAPLR). The non-limiting example of such a regulatory element is a transcription factor binding site. In some embodiments, such a regulatory element is integrated into the STAPLR site near the coding sequence of other one or more regulatory elements and / or transgene. For example, cells can be modified with a DNA molecule disclosed herein, the DNA molecule comprising an exogenous nucleotide sequence, and the exogenous nucleotide sequence comprising a transgene and a transcription factor binding site, wherein the transcription factor that can bind to the transcription factor binding site is expressed by another transgene (e.g., in STAPLR, STEL or another safe harbor site) or ectopically in any part of an endogenous gene or genome.

[0112] In some embodiments, the transgene encodes RNA (e.g., small interfering RNA or microRNA) or a target protein. The target protein (as used herein, including peptides) may be, for example, a globular protein (e.g., albumin, globulin, gluten, prolamin, histone, globin or protamine), a fibrin (e.g., a sclerosing protein, such as collagen, elastin, keratin or silk protein) or an intermediate protein. In some embodiments, the target protein is a complex protein, such as a metalloprotein, a chromoprotein, a glycoprotein, a mucin, a phosphoprotein, a lipoprotein. In some embodiments, the target protein is a therapeutic protein (e.g., a protein that can improve or prevent the symptoms of a disease or condition). Non-limiting examples of therapeutic proteins include proteins that are lacking or defective in genetic diseases such as hemophilia and lysosomal storage diseases, hormones, enzymes, cytokines that regulate immunity, recombinant antigen receptors (e.g., chimeric antigen receptors), antibodies, proteins that regulate the differentiation or activity of modified cells (e.g., transcription factors or proteins that keep cells at M1 or M2 polarity), etc. In some embodiments, the protein of interest is a cell marker, a protein for immune escape, or a safety or kill switch for cell therapy. Examples of proteins of interest are, but are not limited to, SOX10, IL-10, IL-12, CD19t, and ThPOK.

[0113] D. Targeting vector

[0114] The present disclosure provides the targeting vector for the integration of exogenous nucleotide sequence into STAPLR. As used herein, "targeting vector" is a nucleic acid comprising an exogenous nucleotide sequence of interest and a sequence homologous to the endogenous chromosome nucleotide sequence flanking the desired integration position in the genome. These flanking homologous sequences are referred to as "homology arms". The homology arms guide the targeting vector to the specific chromosome position in the genome by the advantage of the homology existing between the homology arms and the corresponding endogenous nucleotide sequences. In some embodiments, the targeting vector is a DNA molecule comprising the nucleotide sequence of interest, and its flank is a 5' nucleotide sequence (left homology arm or homology region) and a 3' nucleotide sequence (right homology arm or homology region), wherein the nucleotide sequence homology of the 5' nucleotide sequence and the 3' nucleotide sequence to the nucleotide sequence flanking the integration site in the cell genome, and the target nucleic acid is integrated into the integration site by homologous recombination mediation.

[0115] The 5' and 3' sequences are sufficiently similar to the endogenous nucleotide sequences targeted for homologous recombination so that if the homology arms are integrated (in whole or in part), they will not adversely affect the genetic environment of the integration (e.g., will not affect the function of adjacent genes). In some embodiments, the homology arms are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence in the targeted STAPLR.

[0116] For example, in some embodiments, the intergenic region between the RPL34 gene and the OSTC gene comprises a nucleotide sequence having at least 80% identity to SEQ ID NO: 1, wherein the function of the intergenic region between the RPL34 gene and the OSTC gene remains intact after integration. In some embodiments, the intergenic region between the RPL34 gene and the OSTC gene comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, such that the function of the intergenic region between the RPL34 gene and the OSTC gene remains intact after integration.The same is true for the intergenic region between the ACTB gene and the FSCN1 gene and its identity to SEQ ID NO: 2, the intergenic region between the AKIRIN1 gene and the NDUFS5 gene and its identity to SEQ ID NO: 3, and the intergenic region between the PRDX1 gene and the AKR1A1 gene and its identity to SEQ ID NO: 4, the intergenic region between the PTGES3 gene and the NACA gene and its identity to SEQ ID NO: 5, the intergenic region between the MLF2 gene and the PTMS gene and its identity to SEQ ID NO: 6, the intergenic region between the RAB13 gene and the RPS27 gene and its identity to SEQ ID NO: 7, the intergenic region between the JTB gene and the RAB13 gene and its identity to SEQ ID NO: 8, the intergenic region between the AKR1A1 gene and the NASP gene and its identity to SEQ ID NO: 9, the intergenic region between the NDUFS5 gene and the MACF1 gene and its identity to SEQ ID NO: 10 The same applies to the intergenic region between the SRSF9 gene and the DYNLL1 gene and its identity to SEQ ID NO:10, the intergenic region between the SRSF9 gene and the DYNLL1 gene and its identity to SEQ ID NO:11, the intergenic region between the MYL6B gene and the MYL6 gene and its identity to SEQ ID NO:12, the intergenic region between the GPX1 gene and the RHOA gene and its identity to SEQ ID NO:13, the intergenic region between the HNRNPA2B1 gene and the CBX3 gene and its identity to SEQ ID NO:14, the intergenic region between the ROMO gene and the RBM39 gene and its identity to SEQ ID NO:15, the intergenic region between the PA2G4 gene and the RPL41 gene and its identity to SEQ ID NO:16, and the intergenic region between the NDUFB10 and RPS2 genes and its identity to SEQ ID NO:97.

[0117] In the methods of the present disclosure, the lengths of the homology arms are different. In some embodiments, each homology arm is independently about at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900 or at least 2000 base pairs long. In some embodiments, each homology arm is independently 50-2000, 50-1500, 100-1900, 150-1800, 200-1700, 250-1600, 300-1500, 350-1400, 400-1300, 450-1200, 500-1100, 550-1000, 600-950, 650-900, 700-850, or 750-800 base pairs in length.

[0118] In the methods of the present disclosure, homology arms (ie, 5' and 3' nucleotide sequences) can be designed to target any position within the disclosed intergenic regions.Homology arms can be designed based on genomic sequences available in sequence databases (eg, NCBI database).

[0119] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 17 necessary for the function of the sequence to remain intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 18 necessary for the function of the sequence to remain intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 17 so that the function of SEQ ID NO: 17 remains intact. In some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 18 so that the function of SEQ ID NO: 18 remains intact.

[0120] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 19 necessary for the function of the sequence to remain intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 20 necessary for the function of the sequence to remain intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 19 to keep the function of SEQ ID NO: 19 intact. Similarly, in some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 20 to keep the function of SEQ ID NO: 20 intact.

[0121] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 21 necessary for the function of the sequence to remain intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 22 necessary for the function of the sequence to remain intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 21 so that the function of SEQ ID NO: 21 remains intact. Similarly, in some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 22 so that the function of SEQ ID NO: 22 remains intact.

[0122] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 23 necessary for the function of the sequence to remain intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 24 necessary for the function of the sequence to remain intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 23 so that the function of SEQ ID NO: 23 remains intact. Similarly, in some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 24 so that the function of SEQ ID NO: 24 remains intact.

[0123] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 93 necessary for the function of the sequence to remain intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 94 necessary for the function of the sequence to remain intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 93 to keep the function of SEQ ID NO: 93 intact. Similarly, in some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 94 to keep the function of SEQ ID NO: 94 intact.

[0124] In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 95 to keep the function of the sequence intact, and the 3' nucleotide sequence comprises a nucleotide sequence sufficiently similar to SEQ ID NO: 96 to keep the function of the sequence intact. In some embodiments, the 5' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 95 to keep the function of SEQ ID NO: 95 intact. Similarly, in some embodiments, the 3' nucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 96 to keep the function of SEQ ID NO: 96 intact.

[0125] In some embodiments, the homology arms fall completely within the targeted STAPLR. In other embodiments, the homology arms may overlap with a portion of an adjacent gene after integration without disrupting its function, and the exogenous sequence is still integrated within the STAPLR.

[0126] In some embodiments, the targeting vector is a circular vector. In some embodiments, the targeting vector is a linear vector. In some embodiments, the targeting vector as provided herein comprises one or more endonuclease targeting sequences, such as, when used in combination with an endonuclease-guide, the vector is linearized. In some embodiments, the targeting vector is a viral vector (e.g., AAV vector, adenoviral vector, lentiviral vector, herpes simplex virus vector) or a plasmid vector.

[0127] The present disclosure provides a STAPLR targeting system comprising a targeting vector herein and a suitable gene editing system, such as those described herein, for incorporating a nucleotide sequence of interest on the targeting vector into STAPLR.

[0128] III. Genetically Modified Mammalian Cells

[0129] Provided herein are genetically modified cells comprising modifications in one or more of the STAPLRs disclosed herein. Targeted mammalian cells for STAPLR integration can be any cell type or cells in any target cell state. For example, cells can be pluripotent cells (e.g., pluripotent stem cells) or differentiated cells. The cells, such as human cells, can be engineered in vitro, in vivo or ex vivo by gene editing methods (e.g., those described herein). Cells can also be non-human cells, such as cells from laboratory animals (e.g., non-human primates, mice, rats and rabbits), farm animals (e.g., cattle and horses) and pets (e.g., dogs and cats).

[0130] A. Stem cells

[0131] In some embodiments, the mammalian cell targeted for modification at its STAPLR is a stem cell, particularly a pluripotent stem cell (PSC), such as an induced pluripotent stem cell (iPSC; such as human iPSC) or an embryonic stem cell (ESC; such as human ESC). The engineered stem cells can then be induced to differentiate into the desired cell type, referred to herein as PSC-derivatives, PSC-derived cells, or PSC-derived cells. Stem cells can be the starting point for potentially generating large numbers of cells of a specific cell type, which are delivered for regenerative medicine in patients with different diseases.

[0132] As used herein, the term "pluripotent" or "pluripotency" refers to the ability of a cell to self-renew and differentiate into cells of any of the three germ layers: endoderm, mesoderm, or ectoderm. "Pluripotent stem cells" or "PSCs" include, for example, ESCs derived from the inner cell mass of a blastocyst or by somatic cell nuclear transfer and iPSCs derived from non-pluripotent cells.

[0133] As used herein, the terms "embryonic stem cells," "ES" cells, and "ESCs" refer to pluripotent stem cells obtained from early embryos. In some embodiments, the term does not include stem cells involving the destruction of human embryos; that is, ESCs are obtained from previously established ESC cell lines.

[0134] The term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell artificially prepared from non-pluripotent cells (e.g., adult somatic cells, partially differentiated cells, or terminally differentiated cells, such as fibroblasts, hematopoietic lineage cells, muscle cells, neurons, epidermal cells, etc.) by introducing or contacting one or more reprogramming factors. Methods for generating iPSCs include, for example, inducing the expression of one or more genes (e.g., POU5F1 / OCT4 (gene ID: 5460) in combination with, but not limited to, SOX2 (gene ID: 6657), KLF4 (gene ID: 9314), c-MYC (gene ID: 4609), NANOG (gene ID: 79923) and / or LIN28 / LIN28A (gene ID: 79727)). Reprogramming factors can be delivered in various ways (e.g., viral, non-viral, RNA, DNA, or protein delivery); alternatively, endogenous genes can be activated by reprogramming non-pluripotent cells into PSCs using, for example, CRISPR tools. See, e.g., WO 2013 / 177133 and WO 2022 / 204567.

[0135] Methods for inducing PSC differentiation into cells of various lineages are known in the art. For example, methods for inducing PSC differentiation into dendritic cells are described in Slukvin et al., JImm. (2006) 176: 2924-32; and Su et al., Clin Cancer Res. (2008) 14 (19): 6207-17; and Tseng et al., Regen Med. (2009) 4 (4): 513-26. Methods for inducing PSC into hematopoietic progenitor cells, myeloid cells and T lymphocytes are described in, for example, Kennedy et al., Cell Rep. (2012) 2: 1722-35.

[0136] Recombinant PSCs can be differentiated into cells suitable for therapeutic use, including cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or their progenitors), ectoderm (e.g., skin, neuronal, or pigment cells, or their progenitors), and mesodermal (e.g., cardiac cells, skeletal muscle cells, erythrocytes, smooth muscle cells, or their progenitors or precursors) lineages.

[0137] In some embodiments, the recombinant PSC is differentiated into cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors or precursors thereof), ectoderm (e.g., skin, neuron, or pigment cell, or progenitors or precursors thereof), or mesodermal (e.g., cardiac cells, skeletal muscle cells, erythrocytes, smooth muscle cells, or progenitors or precursors thereof) lineages.

[0138] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into cardiac cells. In various embodiments, the cardiac cells are cardiac progenitor cells or mature or immature (atrial or ventricular) cardiomyocytes. In other embodiments, the cardiac cells are cardiac endothelial cells or junction cells.

[0139] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into human immune cells, optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, myeloid cells, dendritic cells and / or macrophages / monocytes (e.g., immunosuppressive macrophages) or progenitor cells or precursors.

[0140] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into oligodendrocyte progenitor or precursor cells or oligodendrocytes. In some embodiments, the recombinant PSCs of the present disclosure are differentiated into microglia progenitor or precursor cells, or microglia.

[0141] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into cells of a neural lineage, such as neural crest cells, astrocytes, dopaminergic neuron progenitor cells, dopaminergic neurons, midbrain dopaminergic neuron progenitor cells, midbrain dopaminergic neurons, true midbrain dopamine (DA) neurons, dopaminergic neuron precursor cells, floorplate midbrain progenitor cells, floorplate midbrain DA neurons, or progenitors or precursors thereof.

[0142] In some embodiments, the recombinant PSC of the present disclosure is differentiated into cells of the eye system, such as photoreceptor cells, photoreceptor progenitor cells or precursor cells, retinal pigment epithelial (RPE) cells or their progenitor cells or precursor cells, neural retinal cells or their progenitor cells or precursor cells. In other embodiments, unedited PSC is differentiated into cells of the eye system, and then engineered with the targeting construct of the present disclosure.

[0143] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into microglia or microglial progenitor or precursor cells.

[0144] In a further embodiment, the recombinant PSC of the present disclosure is differentiated into a cell in the human metabolic system, optionally selected from a hepatocyte, a bile duct cell, and a pancreatic β cell, or a progenitor or precursor thereof.

[0145] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into intestinal progenitor or precursor cells or enterocytes.

[0146] B. Differentiated cells

[0147] In other embodiments, the cell to be engineered is a differentiated cell (e.g., a partially or terminally differentiated cell). Partially differentiated cells can be, for example, tissue-specific progenitor cells or stem cells, such as hematopoietic progenitor cells or stem cells, skeletal muscle progenitor cells or stem cells, cardiac progenitor cells or stem cells, neuronal progenitor cells or stem cells, and mesenchymal stem cells.

[0148] Exemplary differentiated cell types that can be engineered at one or more of their STAPLRs include cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neurons, or pigment cells, or progenitors thereof, or precursors thereof), and mesoderm (e.g., cardiac cells, skeletal muscle cells, erythrocytes, smooth muscle cells, or progenitors thereof, or precursors thereof) lineages. Alternatively, PSCs can be differentiated into cells in these lineages and then engineered with the targeting constructs of the present disclosure.

[0149] In some embodiments, cardiac cells are engineered. In some embodiments, the cardiac cells are cardiac progenitor cells or mature or immature (atrial or ventricular) cardiomyocytes. In other embodiments, the cardiac cells are cardiac endothelial cells or junction cells.

[0150] In some embodiments, human immune cells are engineered. Human immune cells are optionally selected from T cells (e.g., CD4+T cells, CD8+T cells, or Treg cells), T cells expressing chimeric antigen receptors (CARs) or recombinant TCRs, regulatory T cells, myeloid cells, dendritic cells, and / or macrophages (e.g., immunosuppressive macrophages) or their progenitor cells or precursors, such as hematopoietic stem cells or progenitor cells.

[0151] In some embodiments, oligodendrocyte progenitor or precursor cells or oligodendrocytes are engineered.

[0152] In some embodiments, a neural lineage cell is engineered. In various embodiments, the neural lineage cell is a neural crest cell, an astrocyte, a dopaminergic neuron progenitor cell, a dopaminergic neuron cell, a midbrain dopaminergic neuron progenitor cell, a midbrain dopaminergic neuron, a true midbrain dopamine (DA) neuron, a dopaminergic neuron precursor cell, a floor plate midbrain progenitor cell, a floor plate midbrain DA neuron, or a progenitor or precursor thereof.

[0153] In some embodiments, cells of the ocular system are engineered. In various embodiments, the cells of the ocular system are photoreceptor cells, photoreceptor progenitors or precursors, retinal pigment epithelial cells or progenitors or precursors thereof, neural retinal cells or progenitors or precursors thereof.

[0154] In further embodiments, microglial cells or microglial progenitor or precursor cells are engineered.

[0155] In further embodiments, cells in the human metabolic system are engineered.In various embodiments, cells in the human metabolic system are optionally selected from hepatocytes, bile duct cells, and pancreatic beta cells, or progenitors or precursors thereof.

[0156] In a further embodiment, intestinal progenitor or precursor cells or intestinal cells are engineered.

[0157] Additional cell types that can be engineered herein to integrate exogenous sequences into STAPLR are, but are not limited to, fibroblasts, adipocytes, muscle cells (e.g., skeletal muscle or smooth muscle cells), osteocytes, myeloid cells, myeloid progenitor cells (e.g., primitive myeloid progenitor cells).

[0158] Cells can be from established cell lines, or they can be primary cells, wherein "primary cells", "primary cell lines" and "primary cultures" are used interchangeably herein, all referring to cells and cell cultures that have been derived from a subject (e.g., a human) and allowed to grow in vitro or ex vivo for a limited number of passages in culture. For example, primary cultures include cultures that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times but are insufficient to undergo a crisis phase. Primary cell lines can be maintained in vitro or ex vivo for less than 10 generations. In some embodiments, cells are autologous in the context of cell therapy. In some embodiments, cells are allogenic in the context of cell therapy.

[0159] Primary cells can be harvested from an individual by any suitable method. For example, leukocytes can be suitably harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most suitably harvested by biopsy.

[0160] Any of the aforementioned differentiated cell types can be differentiated from PSCs prior to engineering them.

[0161] The present disclosure provides a pharmaceutical composition comprising the engineered cells herein and a pharmaceutically acceptable carrier.

[0162] IV. Methods for Identifying STAPLR

[0163] The disclosure also provides methods for identifying STAPLR as a site of safe genome integration in mammalian cells (e.g., human cells). In these methods, the first step is to select a group of cell types for single-cell RNA sequencing ("scRNAseq"). Examples of cell types are, but not limited to, those mentioned herein, including, but not limited to, PSC (e.g., iPSC), cells in the immune system (e.g., T cells, NK cells, dendritic cells, macrophages / monocytes or their hematopoietic progenitor cells), cells in the cardiovascular system (e.g., ventricular cardiomyocytes, knot cells or cardiac progenitor cells), cells in the metabolic system (e.g., hepatocytes and pancreatic beta cells), cells in the central nervous system (e.g., sensory neurons, motor neurons, interneurons, microglia, oligodendrocytes or their progenitor cells), muscle cells (e.g., skeletal muscle cells and smooth muscle cells), cells in adipocytes and eye systems (e.g., retinal pigment epithelial cells and photoreceptor cells).

[0164] The second step is to perform a scRNAseq assay, in which sequencing analysis assigns a unique transcriptome containing transcribed genes to each cell that passes quality criteria. To pass quality criteria, transcriptomes are filtered to exclude those with high rarefaction or absence and those that may originate from more than one cell.

[0165] Next, a prevalence score is assigned to each gene. The prevalence score is based on "1" and represents the fraction of cells containing at least one transcript of a given gene based on the scRNAseq database of the collected data set. In some embodiments, the scRNAseq data set is obtained from PSCs, dopaminergic neurons and / or progenitor cells thereof (e.g., those in various selected differentiation states), microglia and / or progenitor cells thereof (e.g., those in various selected differentiation states), cardiomyocytes and / or progenitor cells thereof (e.g., those in various selected differentiation states), oligodendrocytes and / or progenitor cells thereof (e.g., those in various selected differentiation states), or macrophages and / or progenitor cells thereof (e.g., those in various selected differentiation states).

[0166] After assigning a prevalence score, the location of each gene in the mammalian (eg, human) genome is determined.

[0167] The next step of identifying the STAPLR in the mammalian cell genome is to identify adjacent, non-overlapping genes." non-overlapping genes " refers to that genes are separated from each other by at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1000 base pairs, at least 1500 base pairs, at least 2000 base pairs, at least 2500 base pairs, at least 3000 base pairs, 3500 base pairs, at least 5000 base pairs, at least 10000 base pairs, at least 15000 base pairs or at least 20000 base pairs on either chain. The transcript for calculating the genetic distance for identifying non-overlapping genes can be specified by any genome database, for example RefSeq database and the GENCODE database of NCBI.

[0168] In some cases, different genomic databases contain non-shared gene boundary annotations, which can lead to different calculated genetic distances and opposite conclusions on whether two genes overlap. In this case, if two genes are determined to be non-overlapping by using at least one genomic database, they are considered to be non-overlapping. For example, the downstream flank of MLF2 is its neighboring gene PTMS. As annotated in the NCBI RefSeq database, these genes are non-overlapping with an intergenic distance of approximately 13 kb; however, the GENCODE V38 database reports an MLF2 transcript whose transcription start site is located within the first intron of the PTMS encoded by the opposite strand of the coding pair. In this case, the RefSeq annotation is considered without considering the GENCODE annotation, and the gene pair is classified as non-overlapping.

[0169] Once two or more genes are considered non-overlapping, the proximity scores of non-overlapping gene pairs or regions containing three or more non-overlapping genes are determined. The proximity scores are the product of the individual prevalence scores and reflect the probability of two genes being transcriptionally active in the total scRNAseq dataset. The proximity scores are essentially a ranking of the proximity of transcriptionally active genes.

[0170] The proximity scores are then sorted to obtain a ranking of non-overlapping gene pairs or a ranking of regions containing three or more genes. When the proximity scores are sorted, a pair of genes or a region containing three or more genes with the best proximity score is selected, and the intergenic regions between the genes of the selected pair or region are identified as potential STAPLRs.

[0171] STAPLR can be targeted for safe genetic integration.Then annotate the intergenic region with high ranking adjacent score, so as to design the homology arm for site-specific integration.Usually, the sequence to be avoided in the integration site includes promoter region, enhancer region, CpG island, epigenetic marker (such as, H3K4Me1, H3K4Me3 and H3K27Ac), DNase I hypersensitive peak, conserved region and repeat region.UCSC genome browser (UCSC Genome Browser) can be used with, but not limited to, following gene annotation track (gene annotation track) together: GENCODE V32, RefSeq Genes, GTEx RNA-seq, EPDnewPromoters, ENCODE (transcript, H3K4Me1, H3K4Me3, H3K27Ac and DNase Clusters), GeneHancer, CpG Islands, Conservation 100vertebrates and RepeatMasker.

[0172] When selecting a targetable intergenic subregion, known promoter regions and enhancer regions must be avoided. In addition, conserved regions, repeat regions, epigenetic markers, and DNA enzyme hypersensitive regions are features that should be minimized when selecting a targetable region. In some embodiments, the targetable intergenic subregion comprises a sequence of a CRISPR endonuclease protospacer adjacent motif (PAM) site. The PAM site is a DNA sequence of 2-6 base pairs immediately following the DNA sequence targeted by the Cas (e.g., Cas9 or Cpf1) endonuclease. A short oligonucleotide called a guide RNA (gRNA) is synthesized to perform the function of the tracrRNA-crRNA complex in the CRISPR / Cas gene editing system. gRNA recognizes a gene sequence with a PAM sequence at the 5' or 3' end. Different Cas proteins can recognize different PAMs. For example, Cas9 derived from Streptococcus pyrogenes recognizes 5'-NGG-3' ("N": any nucleobase); Cas9 derived from Staphylococcus aureus recognizes 5'-NNGRR(N)-3'; Cas9 derived from Neisseria meningitidis recognizes 5'-NNNNGATT-3'; Cas9 derived from Campylobacter jejuni recognizes 5'-NNNNRYAC-3' ("Y": pyrimidine); Cas9 derived from Streptococcus thermophilus recognizes 5'-NNAGAAW-3' ("W": A or T); Cas9 derived from Lachnospiraceae recognizes 5'-NNNNGATT-3' ("Y": pyrimidine); bacterium and Acidaminococcus sp. recognize 5'-TTTV-3' ("V": G, A or C); Cas12b derived from Alicyclobacillus acidiphilus recognizes 5'-TTN-3'; and Cas12b v4 derived from Bacillus hisashii recognizes 5'-ATTN-3', 5'-TTTTN-3' and 5'-GTTN-3'.

[0173] Finally, the identified intergenic region can be confirmed to safely support exogenous genetic payloads by inserting a transgene at a targeted position within the intergenic region using a gene editing system. The gene editing system can be, for example, a CRISPR system (e.g., using those of the CRISPR endonucleases disclosed above), a Cre / Lox system, a FLP-FRT system, a TALEN system, a ZFN system, a system utilizing a homing endonuclease, a system producing homologous recombination, or a system utilizing a non-nuclease-dependent viral vector (e.g., a retrovirus, AAV, or a lentiviral vector). Constitutive, inducible, tissue-specific, or lineage-specific promoters can be used to direct the expression of the inserted transgene.

[0174] In some embodiments, the length of the targeted intergenic region is at least 30, 40, 50, 75 or 100 base pairs. In some embodiments, the intergenic region does not include a promoter region or an enhancer region. Although the intergenic region does not include a conserved region, a repeat region, an epigenetic marker and / or a DNA enzyme hypersensitivity region, it may be better, and in some embodiments, the intergenic region may actually include a minimum amount of conserved regions, repeat regions, epigenetic markers and / or enzyme hypersensitivity regions. For example, in some embodiments, the intergenic region does not include CpG islands, H3K4Me1 epigenetic markers, H3K4Me3 epigenetic markers, H3K27Ac epigenetic markers, DNase I hypersensitive regions, conserved regions or repeat regions. However, in some embodiments, the intergenic region may include CpG islands, H3K4Me1 epigenetic markers, H3K4Me3 epigenetic markers, H3K27Ac epigenetic markers, DNAseI hypersensitive regions, conserved regions or repeat regions. The amount of conserved regions, repetitive regions, epigenetic marks, and / or DNA enzyme hypersensitive regions allowed depends on various factors. These factors include, for example, the size of the intergenic region; the size of the conserved, repetitive and / or hypersensitive regions or epigenetic marks; the presence of gRNA binding sites; or the feasibility of synthesizing 5' and 3' homology arms for targeting.

[0175] After genomic integration, the transcription level of the integrated transgene is measured, and when the integrated transgene shows persistent transcription (or shows persistent transcription when the inducible promoter regulating the transgene is induced), the intergenic region between the selected pair or within the selected region is identified as STAPLR.

[0176] Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to the methods and materials described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the present specification, including definitions, shall prevail. Further, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout the present specification and embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" shall be understood to indicate that the integer or integer group is included, but any other integer or integer group is not excluded. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although many documents are cited herein, the citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, when applied to one or more target values, the term "approximately" or "about" refers to a value similar to the reference value. In some embodiments, unless otherwise stated or obvious from the context, the term refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) of the reference value.

[0177] According to the present disclosure, the back-references in the dependent claims are meant as shorthand for the direct and unambiguous disclosure of each and every combination of claims indicated by the back-reference. Further, the headings herein are created for organizational convenience and are not intended to limit the scope of the claimed invention in any way.

[0178] In order to better understand the present invention, the following examples are given. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0179] Example

[0180] In order to better understand the present invention, the following examples are given. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0181] Example 1: Design for STAPLR targeting

[0182] Selection of gRNA

[0183] We used CRISPOR to identify Cas9-based gRNAs targeting near the midpoint region where the STAPLR construct homology arms flank the expected site of transgene integration. We excluded gRNAs that were completely off-target in the genome. We minimized the use of gRNAs with a maximum of 3 bp off-target mismatches. Three gRNAs were selected for each STAPLR site, as shown in Table 2.

[0184] Table 2. gRNAs used to target selected STAPLR sites

[0185]

[0186]

[0187] *Preferred embodiment

[0188] A list of other Cas9- and Cpf1-based gRNAs for STAPLR targeting is listed in Table 3.

[0189] Table 3. Other gRNAs targeting STAPLR

[0190]

[0191]

[0192] For each STAPLR site, human iPSCs were nucleofected with each individual gRNA complexed with the Cas9 nuclease in the form of a ribonucleoprotein (RNP). Three days later, the nucleofected cells were harvested, genomic DNA was extracted, and PCR amplification of the genomic region flanking the expected cleavage site was performed. The purified PCR products were sequenced, and the overall cleavage efficiency ( ) of the sequencing data was analyzed by Synthego's ICE Analysis Tool (available on Synthego's website). Figure 1 ). gRNAs were considered efficient when showing greater than 50% indel editing.

[0193] The data show that there is at least one efficient gRNA per STAPLR site (>50% indel editing). The gRNA with the greatest overall cutting efficiency was selected for future experiments to integrate transgenes at the STAPLR site.

[0194] Design of STAPLR homology arms

[0195] Generate a list of neighboring genes composed of all highly expressed genes. Filter the list to remove the gene pairs containing at least one known tumor suppressor gene or oncogene. First, subtract the gene pairs with a gene spacing distance less than 5kb between them. However, the gene pairs with only about 100 base gene spacing distances between the flanking genes can also be annotated and tested. Promoter regions, enhancer regions, CpG islands and regions containing epigenetic marks are avoided in the design. Subregions that avoid regulatory elements and can be synthesized in donor plasmids are classified as potential homology arm regions and used as the basis for gRNA searches (Table 4).

[0196] Table 4. Parameters used to select STAPLR

[0197] STAPLR locus parameters Sites flanked by adjacent highly expressed genes Neither of the two flanking genes is a known tumor suppressor or oncogene The intergenic distance between flanking genes is >50 bases Sites avoid annotated regulatory elements and repetitive regions Sites allow synthesis of homology arms and cloning into donor constructs The loci have available CRISPR gRNAs with predicted high efficacy and low off-target numbers

[0198] After selecting a gRNA with a predicted high efficiency, the homology arm sequence was finally determined to be the center of the selected gRNA within the 800 bp left homology arm and 800 bp right homology arm flanking the expected site of transgene integration. Table 5 shows the intergenic distance in base pairs between two adjacent genes of each exemplary STAPLR site, as well as the coordinates of each set of STAPLR left and right homology arms based on the hg38 human reference genome. Gene distances were calculated using the RefSeq database of NCBI.

[0199] Table 5. Intergenic distances between STAPLR neighboring genes and STAPLR homology arm coordinates

[0200]

[0201] The sequences of the left and right homology arms of the targeting construct based on the hg38 human reference genome are shown in the table below.

[0202] Table 6. STAPLR left homology arm and right homology arm

[0203]

[0204]

[0205]

[0206]

[0207] Example 2: Testing inducibility and transgene expression at STAPLR in pooled populations of targeted iPSCs

[0208] To test the inducibility and robustness of transgene expression at each of the four annotated STAPLR loci (PRDX1-AKR1A1 (site 1), ACTB-FSCN1, RPL34-OSTC, AKIRIN1-NDUFS5), the two-component doxycycline-inducible rtTA / TRE system Tet-On 3G from Clontech / TakaRa (described in U.S. Pat. No. 9,127,283, which is incorporated herein by reference in its entirety) was used. For the constitutive component, Tet-On 3GrtTA (reverse tetracycline transactivator) was used by the inventors' "Sustained Transgene Expression Loci" (STEL) method described in WO 2021 / 072329 (which is incorporated herein by reference in its entirety) ( Figure 2 ), expressed biallelically from the GAPDH locus.

[0209] To test the inducibility of the transgene from the STAPLR locus, the TRE3G promoter was used to test the expression of the eGFP cargo. A Kozak sequence was included to enable translation initiation, and an SV40 PolyA sequence was added to enable transcription termination. In the presence of doxycycline, the rtTA protein binds and activates the tetracycline response element (TRE) minimal promoter ( Figure 3 ). For each STAPLR locus, the parental iPSC line with biallelic rtTA integration in GAPDH (GAPDH::rtTA iPSCs) was nucleofected with the selected efficient RNP and the corresponding STAPLR targeting construct (STAPLR left homology arm-TRE3G promoter-eGFP-SV40-STAPLR right homology arm). Starting from the first day after nucleofection, the cell pool that received STAPLR RNP and STAPLR targeting construct was cultured with medium containing 2 μg / ml doxycycline ( Figure 4 ) and continued until day 7 after nuclear transfection ( Figure 5 ) to induce GFP expression. The parental rtTA iPSC line was also given 2 μg / ml doxycycline culture medium as a control. GFP expression was monitored over the course of a week by fluorescence microscopy. As cells were treated with doxycycline for longer periods of time, an increase in GFP intensity was observed. Preliminary testing of this rtTA / TRE-based transgenic expression system in STAPLR showed robust inducibility and expression of GFP in a combined population of STAPLR site-targeted iPSCs.

[0210] Example 3: Testing inducibility and transgene expression at STAPLR in clonal populations of targeted iPSCs

[0211] Parental GAPDH::rtTAiPSCs were nuclear transfected with RNP and STAPLR targeting constructs at each of the four STAPLR sites, and then the pooled population of each STAPLR targeting iPSC was plated at clonal density. Single clones were selected and screened by PCR across the junction of the left and right homology arms to confirm the precise integration of TRE3G-eGFP-SV40 at each of the four STAPLR sites. Targeted iPSC clones were amplified and treated with a culture medium containing 0.1 μg / ml to 5 μg / ml of doxycycline for 0 to 68 hours. During this time course of GFP induction, cells were collected during the time course of 0, 3, 8, 24, 48 and 68 hours, and flow cytometry analysis was performed ( Figure 6 ). The results showed that maximum GFP induction from all four STAPLR sites was seen from the administration of 0.1 μg / ml doxycycline and 48 hours after doxycycline administration. The maximum expression levels of GFP from the STAPLR sites were different, with the PRDX1-AKR1A1 site showing the highest expression of GFP in doxycycline-induced iPSCs. One clone-derived line derived from each STAPLR targeted site and a wild-type, unedited iPSC control line were then treated with medium containing 2 μg / ml doxycycline for 72 hours ( Figure 7 The AKIRIN1-NDUFS5 STAPLR line showed a slight delay in GFP induction, so treatment was increased to 6 days with medium containing 2 μg / ml doxycycline ( Figure 7 ). The results showed that all four treated STAPLR-targeted iPSC lines could induce high levels of GFP expression, with the PRDX1-AKR1A1 locus again demonstrating the highest expression of GFP in doxycycline-induced iPSCs, while the wild-type, unedited, doxycycline-treated iPSC control line did not express GFP. In all cases, cells that did not receive doxycycline treatment did not express GFP.

[0212] Example 4: Testing inducibility and transgene expression at STAPLR in iPSC-derived myeloid progenitor cells

[0213] The clonally derived STAPLR iPSC lines were differentiated into myeloid progenitors to demonstrate that transgene integration at STAPLR maintains sustained transgene expression in differentiated iPSCs (Douvaras et al., Stem Cell Reports (2017) 8(6): 1516-24). On day 12 of differentiation, 2 μg / ml doxycycline was added to each STAPLR targeted clonal line and supplemented with doxycycline daily for three days. On day 15 of differentiation, adherent myeloid progenitors were harvested for flow cytometric analysis of GFP induction. Three of the four TRE-eGFP-SV40 STAPLR lines (PRDX1-AKR1A1, ACTB-FSCN1, RPL34-OSTC) showed efficient GFP induction in heterogeneous adherent myeloid progenitors compared to differentiated cells that did not receive doxycycline ( Figure 8 ). A wild-type unedited iPSC control line differentiated using the same protocol and similarly treated with doxycycline showed no induction of GFP. One of the TRE-eGFP-SV40 STAPLR lines (AKIRIN1-NDUFS5) showed delayed GFP induction under fluorescence microscopy. The cell line was supplemented with doxycycline for an additional three days, and adherent myeloid progenitors were harvested on day 18 of differentiation for flow cytometry analysis. Figure 8 Shown is the bimodal GFP induction observed in myeloid progenitor cells harvested at differentiation day 18. In all cases, cells not treated with doxycycline did not express GFP.

[0214] The STAPLR targeted lines were further differentiated after 30 days to the extent that non-adherent myeloid progenitors could be collected in suspension culture. 2 μg / ml doxycycline was added for 6 days and non-adherent myeloid progenitors were collected for flow cytometric analysis of GFP induction. All four TRE-eGFP-SV40 STAPLR lines cultured for more than 30 days demonstrated efficient differentiation into triple-positive myeloid progenitors as defined by >80% co-expression of cell surface markers CD45, CD14, and CX3CR1 ( Fig. 9 The doxycycline-treated STAPLR line also exhibited efficient GFP induction in heterogeneous non-adherent myeloid progenitor cells, with some variation in maximal GFP expression levels compared to the doxycycline-treated wild-type unedited control line ( Fig.10 ). This data demonstrates that integration of the transgene at all four STAPLR sites allows for sustained expression of the transgene under the control of the external promoter during and after differentiation into myeloid progenitors.

[0215] Example 5: Derivation of a human induced pluripotent stem cell line expressing CD19t-IL12 at the PRDX1-AKR1A1 STAPLR locus

[0216] The parental iPSC line with biallelic rtTA integration in GAPDH (GAPDH::rtTA iPSCs) was transfected with the selected efficient RNP transfection PRDX1-AKR1A1 STAPLR locus (site 1) and a STAPLR targeting construct containing a CD19t-IL12 cassette (flanked by the PRDX1-AKR1A1 left homology arm and the PRDX1-AKR1A1 right homology arm) driven by a doxycycline-inducible promoter (TRE3G). CD19t was included here as a non-biological functional cargo; it was used as an epitope tag for surrogate detection of IL-12 transgene integration by flow cytometry. Two different gRNAs and their corresponding nucleases were used to target the PRDX1-AKR1A1 STAPLR locus. A Cpf1-based guide RNA with the sequence 5′-GAGACTGGTTCTTGCAGCACT-3′ (SEQ ID NO: 83) or a Cas9-based guide RNA with the sequence 5′-CTTGCAGCACTGCCTAGGCT-3′ (SEQ ID NO: 71) was selected to generate clonal lines. From the GAPDH locus, GAPDH::rtTA constitutively expresses the reverse tetracycline transactivator (rtTA). In the presence of doxycycline, rtTA binds to the TRE3G promoter and induces the expression of CD19t and IL-12 driven by the TRE3G promoter ( Fig.11 ).

[0217] Single cell suspensions of GAPDH::rtTA iPSCs were prepared for transfection with Cpf1 or Cas9gRNA RNP complexes and the PRDX1-AKR1A1-targeted pTRE3G-CD19t-IL-12 DNA donor template. Two days after transfection, cells were treated with doxycycline (2 μg / ml) for 48 h to induce CD19t-IL12 expression and analyzed by live cell imaging using AF488-conjugated anti-CD19t antibody staining ( Fig.12 , Figures A and B). The cells were then dissociated and plated at single cell clonal density. Four days after clonal density plating, growing clones were treated with 2 μg / ml doxycycline for 48 hours to induce CD19t-IL-12 expression. After 48 hours of doxycycline treatment, clones were analyzed using live cell imaging using AF488-conjugated Ab against CD19t. CD19t-positive colonies were identified ( Fig.12 , Panels A and B, labeled under “Clone density”).

[0218] The data demonstrate that integration of the CD19t-IL-12 expression cassette at the PRDX1-AKR1A1 STAPLR locus allows for sustained expression of the transgene under the control of an external promoter in both the total pooled and clonal populations of STAPLR-targeted iPSCs following treatment with doxycycline.

[0219] Example 6: Inducing reporter transgene expression at different sites within the STAPLR intergenic region in targeted iPSCs

[0220] To test the inducibility and robustness of transgene expression from two alternative sites within the PRDX1-AKR1A1 intergenic region (PRDX1-AKR1A1 site 2 and site 3), we again utilized the two-component doxycycline-inducible rtTA / TRE system. The TRE3G promoter was used to test expression of the EGFP cargo. Based on the initial PRDX1-AKR1A1 targeting construct design, a Kozak sequence was included to enable translation initiation and an SV40 polyA sequence was added to enable translation termination. In the presence of doxycycline, the rtTA protein binds and activates the TRE minimal promoter. Parental iPSC lines with biallelic rtTA integration in GAPDH (GAPDH::rtTA iPSCs) were nucleofected with the selected high-efficiency RNPs and the corresponding PRDX1-AKR1A1 targeting construct (for site 2 or site 3). Three different gRNAs were tested for PRDX1-AKR1A1 site 2 (SEQ ID NOs: 87-89), and three different gRNAs were tested for PRDX1-AKR1A1 site 3 (SEQ ID NOs: 90-92). Pools of cells that received both PRDX1-AKR1A1 site 2 or site 3 RNPs and targeting constructs were isolated from the day after nucleofection (site 2; Fig.13 ) or day 1 (site 3, Fig.14 ) were cultured with medium containing 2 μg / ml doxycycline and continued up to day 7 after nucleofection ( Fig.15 and Fig.16) to induce GFP expression. GFP expression was monitored by fluorescence microscopy or flow cytometry over the course of 7 days. Both PRDX1-AKR1A1 site 2 and PRDX1-AKR1A1 site 3 induce GFP expression. All three gRNAs tested for each site show differences in construct targeting efficiency (peaks of different sizes are seen in the flow cytometry histogram), but after adding doxycycline, all gRNAs are able to induce GFP expression to similar high intensities (similar logarithmic levels of expression). The peak observed near 10^6 represents edited cells expressing high levels of GFP, while the peak observed near 10^4 represents transient GFP expressed by a targeting construct that has never been integrated. The data show that multiple sites within the PRDX1-AKR1A1 intergenic region allow robust inducibility and expression of GFP in the merged population of iPSCs targeting the STAPLR site.

Claims

1. A genetically modified mammalian cell comprising an exogenous nucleotide sequence integrated into a persistent transcriptionally active payload region (STAPLR) in the cell genome, wherein the STAPLR is selected from: The intergenic region between the RPL34 gene and the OSTC gene; The intergenic region between the ACTB gene and the FSCN1 gene; the intergenic region between the AKIRIN1 gene and the NDUFS5 gene; the intergenic region between the PRDX1 gene and the AKR1A1 gene; The intergenic region between the PTGES3 gene and the NACA gene; The intergenic region between the MLF2 gene and the PTMS gene; the intergenic region between the RAB13 gene and the RPS27 gene; the intergenic region between the JTB gene and the RAB13 gene; the intergenic region between the AKR1A1 gene and the NASP gene; The intergenic region between the NDUFS5 gene and the MACF1 gene; the intergenic region between the SRSF9 gene and the DYNLL1 gene; the intergenic region between the MYL6B gene and the MYL6 gene; the intergenic region between the GPX1 gene and the RHOA gene; the intergenic region between the HNRNPA2B1 gene and the CBX3 gene; the intergenic region between the ROMO gene and the RBM39 gene; the intergenic region between the PA2G4 gene and the RPL41 gene; and The intergenic region between the NDUFB10 and RPS2 genes.

2. A method for modifying a mammalian cell, comprising integrating an exogenous nucleotide sequence in a persistent transcriptionally active payload region (STAPLR) in the cell genome, wherein the STAPLR is selected from: The intergenic region between the RPL34 gene and the OSTC gene; The intergenic region between the ACTB gene and the FSCN1 gene; the intergenic region between the AKIRIN1 gene and the NDUFS5 gene; the intergenic region between the PRDX1 gene and the AKR1A1 gene; The intergenic region between the PTGES3 gene and the NACA gene; The intergenic region between the MLF2 gene and the PTMS gene; the intergenic region between the RAB13 gene and the RPS27 gene; the intergenic region between the JTB gene and the RAB13 gene; the intergenic region between the AKR1A1 gene and the NASP gene; The intergenic region between the NDUFS5 gene and the MACF1 gene; the intergenic region between the SRSF9 gene and the DYNLL1 gene; the intergenic region between the MYL6B gene and the MYL6 gene; the intergenic region between the GPX1 gene and the RHOA gene; the intergenic region between the HNRNPA2B1 gene and the CBX3 gene; the intergenic region between the ROMO gene and the RBM39 gene; the intergenic region between the PA2G4 gene and the RPL41 gene; and The intergenic region between the NDUFB10 and RPS2 genes.

3. The method according to claim 2, wherein the integration step is performed by using the following technology: CRISPR / Cas system; Cre / Lox system; FLP-FRT system; TALEN system; ZFN system; homing endonuclease; random integration; homologous recombination; transposase; or a non-nuclease-dependent viral vector, optionally selected from a retroviral vector, an adeno-associated virus (AAV) vector and a lentiviral vector.

4. The method according to claim 2, wherein the integrating step is performed by using a CRISPR / Cas system comprising a guide RNA, and wherein The STAPLR is a gene spacer between the RPL34 gene and the OSTC gene, and the gRNA is selected from SEQ ID NOs: 25-32, The STAPLR is a gene spacer between the ACTB gene and the FSCN1 gene, and the gRNA is selected from SEQ ID NOs: 33-54, The STAPLR is a gene spacer between the AKIRIN1 gene and the NDUFS5 gene, and the gRNA is selected from SEQ ID NOs: 55-70, or The STAPLR is the intergenic region between the PRDX1 gene and the AKR1A1 gene, and the gRNA is selected from SEQ ID NOs: 71-92.

5. The method of claim 3 or 4, wherein the CRISPR / Cas system comprises a gRNA-dependent nuclease of type I, type II, type III, type IV or type V, or a variant thereof.

6. The method according to claim 3 or 4, wherein the CRISPR / Cas system comprises a gRNA-dependent nuclease selected from the group consisting of Cas9, Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas12, Cas13, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, C sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, CasX, CasY, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, CasPhi, MAD7 and Csf4.

7. A DNA molecule comprising a nucleotide sequence of interest flanked by a 5' homology region (HR) and a 3' HR, wherein the 5' and 3' HR are at least 95% homologous to a first genomic region (GR) and a second GR in a persistent transcriptionally active payload region (STAPLR) in a mammalian cell genome, respectively, wherein the STAPLR is selected from: The intergenic region between the RPL34 gene and the OSTC gene; The intergenic region between the ACTB gene and the FSCN1 gene; the intergenic region between the AKIRIN1 gene and the NDUFS5 gene; the intergenic region between the PRDX1 gene and the AKR1A1 gene; The intergenic region between the PTGES3 gene and the NACA gene; The intergenic region between the MLF2 gene and the PTMS gene; the intergenic region between the RAB13 gene and the RPS27 gene; the intergenic region between the JTB gene and the RAB13 gene; the intergenic region between the AKR1A1 gene and the NASP gene; The intergenic region between the NDUFS5 gene and the MACF1 gene; the intergenic region between the SRSF9 gene and the DYNLL1 gene; the intergenic region between the MYL6B gene and the MYL6 gene; the intergenic region between the GPX1 gene and the RHOA gene; the intergenic region between the HNRNPA2B1 gene and the CBX3 gene; the intergenic region between the ROMO gene and the RBM39 gene; the intergenic region between the PA2G4 gene and the RPL41 gene; and The intergenic region between the NDUFB10 and RPS2 genes.

8. The DNA molecule of claim 7, wherein each 5' and 3' HR is independently about at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900 or at least 2000 base pairs in length; or between 50 and 1500 base pairs in length.

9. The DNA molecule according to claim 7 or 8, wherein the 5' and 3' HR are at least 95% homologous to the following sequences respectively: SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 93 and 94, or SEQ ID NOs:95 and 96.

10. The cell, method or DNA molecule according to any one of claims 1 to 9, wherein: the intergenic region between the RPL34 gene and the OSTC gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1; the intergenic region between the ACTB gene and the FSCN1 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2; the intergenic region between the AKIRIN1 gene and the NDUFS5 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3; the intergenic region between the PRDX1 gene and the AKR1A1 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:4; the intergenic region between the PTGES3 gene and the NACA gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:5; the intergenic region between the MLF2 gene and the PTMS gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 6; the intergenic region between the RAB13 gene and the RPS27 gene comprises a nucleotide sequence at least 95% identical to SEQ ID NO:7; the intergenic region between the JTB gene and the RAB13 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 8; the intergenic region between the AKR1A1 gene and the NASP gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:9; the intergenic region between the NDUFS5 gene and the MACF1 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 10; the intergenic region between the SRSF9 gene and the DYNLL1 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 11; the intergenic region between the MYL6B gene and the MYL6 gene comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 12; the intergenic region between the GPX1 gene and the RHOA gene comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 13; the intergenic region between the HNRNPA2B1 gene and the CBX3 gene comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 14; the intergenic region between the ROMO gene and the RBM39 gene comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 15; The intergenic region between the PA2G4 gene and the RPL41 gene comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; and / or The intergenic region between the NDUFB10 and RPS2 genes comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

97.

11. The cell, method or DNA molecule according to any one of claims 1 to 10, wherein the exogenous nucleotide sequence or the nucleotide sequence of interest comprises a transgene, optionally wherein the transgene comprises a constitutive or inducible promoter.

12. The cell, method or DNA molecule of claim 11, wherein the transgene encodes a therapeutic protein, optionally a protein that is deficient or defective in a genetic disease, a cytokine, or a recombinant antigen receptor; Cell markers; or Proteins that regulate the differentiation state or activity of cells; Optionally, wherein the transgene encodes SOX10, IL-10, IL-12, CD19t or ThPOK.

13. The cell, method or DNA molecule of any one of claims 1-12, wherein the cell is a human cell.

14. The cell, method or DNA molecule of any one of claims 1-13, wherein the cell is a pluripotent stem cell (PSC), optionally an induced PSC (iPSC).

15. The cell, method or DNA molecule according to any one of claims 1 to 13, wherein the cell is: a) a cell of the immune system, optionally a T cell, a natural killer cell, a dendritic cell, a macrophage / monocyte or a hematopoietic progenitor thereof; b) cells in the cardiovascular system, optionally ventricular cardiomyocytes, nodal cells or cardiac progenitor cells; c) a cell in a metabolic system, optionally a hepatocyte, a pancreatic β cell or a bile duct cell; d) a cell in the central nervous system, optionally a sensory neuron, a motor neuron, an interneuron, a microglia, an oligodendrocyte or a progenitor cell thereof; e) muscle cells, optionally skeletal muscle cells or smooth muscle cells; f) fat cells; or g) A cell in the visual system, optionally a retinal pigment epithelial cell, a photoreceptor cell, or a photoreceptor precursor cell.

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