Generation of landing pad cell lines

Abstract

The present disclosure provides methods of generating landing pad cells for targeted gene integration comprising integrating a landing pad plasmid into the genome of a parent cell at a targeted integration site, for example, using homologous recombination. In one aspect, two site-specific recombination sites (SSRSs) are flanked by a polynucleotide sequence; and two homologous recombination sites are located at the 5'and 3 'ends of the SSRS. Two homologous recombination sites of the landing pad plasmid can be recombined with corresponding homologous recombination sites on the parental plasmid, so that the landing pad plasmid is integrated at an internal position in the parental plasmid. The disclosed methods allow for the generation of high expression cell lines by identifying hotspots for targeted integration in thermal cell lines. The present disclosure also provides cells for generating landing pad cells of the disclosure, as well as kits comprising cells and / or agents for generating landing pad cells of the disclosure. Novel hotspots for targeted integration are also provided.

Description

Reference to a sequence listing submitted electronically via EFS-WEB

[0001] The contents of the electronically submitted Sequence Listing (Name: 3338_196PC02_Seqlisting_ST26.txt, Size: 45,039,473 bytes; and Creation Date: December 27, 2022) are incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure provides methods for generating landing pad cells suitable for targeted gene integration. Background Art

[0003] Historically, cell lines were prepared by transfecting cells with expression plasmid DNA (usually linearized) that was integrated into the cell genome in a substantially random manner. Since the plasmid provides a selective advantage (such as drug resistance or auxotrophic complementation), only those cells with the expression plasmid survive. It is desirable to minimize the time spent preparing a cell line expressing a biological product of interest and to increase the predictability of such preparation while maintaining acceptable performance levels, such as high titer in a bioreactor, post-translational modifications, expression stability, cell density, and viability (to name just a few parameters).

[0004] One way to achieve this goal is to use a technique called targeted integration (TI) during cell line development, in which one or more expression cassettes or expression plasmids are inserted into the same locus of the cell line. The targeted locus is also referred to as a landing pad, and a cell line with a landing pad is referred to as a landing pad cell line. One or more expression cassettes or one or more expression plasmids can be integrated into the landing pad by the following methods: site-directed recombination using Cre / Lox technology, sometimes referred to as recombination-mediated cassette exchange (RMCE) or site-specific recombination (SSR); or homologous recombination stimulated by regularly spaced clustered short palindromic repeats (CRISPR), TALEN or other such site-specific nucleases.

[0005] The main challenge of TI is to identify the cell line and locus to use. The cell line itself must be able to perform well, and the landing pad needs to be in a locus (a "hotspot") where high transcription occurs and the transcription of the biologic is not silenced (such as by epigenetic modifications). While the TI landing pad host cell line should have a "hotspot" in the chromosome for high expression, it should be understood that this "hotspot" needs to support the environment of a "hot cell" for all the intermediate steps required for high protein expression of the biologic. The ability to generate and identify landing pad cell lines is very difficult, in part due to variability caused by the inherent plasticity of the host cell genome.

[0006] Therefore, efficient methods to generate landing pad cells capable of reliable and reproducible protein expression are needed. Summary of the invention

[0007] The present disclosure provides a method for selecting parental cells suitable for developing landing pad cell lines, the method comprising: (i) screening and selecting cell lines with high expression titers of a gene of interest (GOI); and (ii) further screening the cells of (i) and selecting cells with low copy numbers of a parental plasmid comprising a nucleic acid encoding the GOI, wherein the copy number is one or two. In some aspects, the parental plasmid comprises two site-specific recombination sites (SSRS), one SSRS, or no SSRS.

[0008] The present disclosure also provides a method for selecting landing pad cells, the method comprising: (i) screening a parental cell line for the loss of the parental plasmid or a portion thereof, and selecting cells having such a loss (deletion); and (ii) further screening the cells of (i) for the presence of a landing pad, and selecting cells in which a landing pad is present.

[0009] Also provided is a method for selecting landing pad cells, the method comprising: (i) screening for loss of at least one parental plasmid or a portion thereof in a parental cell line, and selecting cells having such loss (deletion); and (ii) further screening the cells of (i) for the presence of at least one landing pad, and selecting cells in which a landing pad is present. In some aspects, the method further comprises screening the landing pad sequence in the landing pad cells for a feature selected from the following: (i) presence or absence of low complexity regions or high complexity regions; (ii) presence or absence of retrotransposon sequences; (iii) presence or absence of Alu repeat sequences; (iv) presence or absence of long interspersed nuclear elements (LINEs); (v) presence or absence of CpG islands; (vi) levels of cytosine methylation; (vii) levels of histone acetylation; (viii) presence or absence of active transcription; and (ix) any combination thereof.

[0010] Also provided is a method of generating a landing pad cell, the method comprising (i) causing a deletion of at least one parent plasmid or a portion thereof comprising a first GOI in a parent cell line, and (ii) after the at least one deletion, introducing a landing pad plasmid or a portion thereof comprising a landing pad into the cell. In some aspects, the landing pad plasmid or a portion thereof comprising a landing pad is inserted at the site of the deletion of (i). In some aspects, the landing pad plasmid or a portion thereof comprising a landing pad is inserted at a site that is not a deletion of (i).

[0011] The present disclosure also provides a method of generating a landing pad cell, the method comprising integrating a landing pad plasmid into the genome of a parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is located in the parent plasmid, wherein each landing pad plasmid comprises (1) a polynucleotide sequence comprising a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (2) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1); and (3) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2), which are homologous to corresponding homologous recombination sites in the parent plasmid; and wherein the homologous recombination sites of the landing pad plasmid recombine with corresponding homologous recombination sites of the parent plasmid, thereby integrating the landing pad plasmid at an internal position within the parent plasmid inserted into the genomic DNA of the parent cell. In some aspects, the parent plasmid is located in more than one genomic locus.

[0012] The present disclosure also provides a method for identifying a landing pad cell line, the method comprising (1) removing at least a portion of a first GOI from a parent plasmid integrated into a genomic sequence of a parent cell; (2) integrating a landing pad plasmid at an alternative genomic locus; (3) screening a library of candidate cells comprising at least one copy of the landing pad plasmid integrated at at least one alternative genomic locus, wherein the candidate cell line is evaluated for one or more of the following properties: (a) cell titer exceeding a predetermined threshold level; (b) landing pad plasmid or landing pad copy number at a predetermined value; (c) RNA expression level exceeding a predetermined threshold level, (d) multiple plasmid copies, if present, having a specific plasmid configuration; (e) at least a portion of the first GOI of the parent plasmid is deleted; and (f) the presence of at least one landing pad with a functional SSRS. In some aspects, the parent cell is a historical cell line. In some aspects, the library of candidate cells is a library generated by randomly integrating the landing pad sequence at multiple locations in the genome of the parent cell. In some aspects, the method selects hot cells having the landing pad sequence integrated in a hot spot. In some aspects, the parental cell line is a CHO cell line.

[0013] The present disclosure also provides a method for generating an expression cell, the method comprising integrating a second GOI plasmid into the genome of a landing pad cell by using a site-specific recombinase recombination according to any one of the methods disclosed above, wherein the resulting expression plasmid comprises (1) a polynucleotide sequence containing a nucleic acid encoding a second GOI; and (2) two SSRs flanking the polynucleotide of (1); wherein the site-specific recombination site of the landing pad plasmid is recombined with the corresponding site-specific recombination site of the second GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

[0014] Also disclosed is a method for generating an expressing cell, the method comprising: (a) integrating a landing pad plasmid or a portion thereof into the genome of a parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is located in the parent plasmid, wherein each landing pad plasmid or a portion thereof comprises (1a) a polynucleotide sequence containing a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2a) two SSRS flanking the polynucleotide sequence of (1a); and (3a) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2a), which are homologous to corresponding homologous recombination sites in the parent plasmid; and wherein the homologous recombination sites of the landing pad plasmid or a portion thereof recombine with corresponding homologous recombination sites of the parent plasmid in a landing pad at a different genomic locus, thereby integrating the landing pad plasmid or a portion thereof at an internal position within the landing pad at the different genomic locus in the genomic DNA of the parent cell; and (b) integrating a second GOI plasmid into the genome of the landing pad cell using a site-specific recombinase recombination enzyme, wherein the expression plasmid comprises (1b) a polynucleotide sequence containing a nucleic acid encoding a GOI; and (2b) two SSRs flanking the polynucleotide of (1b); wherein the site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

[0015] Also provided is a method of generating landing pad cells, the method comprising: (a) removing at least a portion of a parent plasmid from a first hotspot location in a parent cell line; and (b) integrating a landing pad plasmid into a second hotspot location in the genome of the parental cell at a targeted integration site using homologous recombination or random integration, wherein the sequence targeted for homologous recombination or random integration is present in the landing pad plasmid, wherein each landing pad plasmid comprises (1b) a polynucleotide sequence containing a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (2b) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1b); and (3b) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2b), which are homologous to corresponding homologous recombination sites in the genome of the parental cell line.

[0016] Also provided is a method for producing an expressing cell, the method comprising: (a) removing a parent plasmid or a portion thereof from a first hotspot location in a parent cell line, (b) integrating a landing pad plasmid into a second hotspot location in the genome of the parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is present in the parent cell line, wherein each landing pad plasmid comprises (1b) a polynucleotide sequence comprising a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (2b) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1b); and (3b) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2b) that are homologous to corresponding homologous recombination sites in the parent cell line, wherein the homologous recombination sites of the landing pad plasmid recombines with the corresponding homologous recombination sites of the parent cell line, thereby integrating the landing pad plasmid at an internal location within the genomic DNA of the parent cell; and (c) integrating the GOI plasmid into the genome of the landing pad cell by recombination using a site-specific recombinase, wherein the expression plasmid comprises (1c) a polynucleotide sequence containing a nucleic acid encoding a first GOI; and (2c) two SSRs flanking the polynucleotide of (1c); wherein the site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

[0017] In some aspects of the methods disclosed above, the landing pad cells comprise a plasmid having a topology corresponding to: CG1 / -[P1]-[P2]-[SSRS]-[M]-[SSRS]-[P2]-[P1]- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-[P2]-[P1]- / CG2; CG1 / -[P1]- / CG2; CG1 / -([P1]-([P2])n-[P1])- / CG2; -[P1]-[P2]-[P1]-; or -([P1]-([P2])n-[P1])- in: CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; and [SSRS] is site-specific recombination site (SSRS). n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0018] In some aspects of the methods disclosed above, the topology of the plasmid integrated in the expression cell corresponds to the following description: CG1 / -[P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[P3]-[SSRS]-[P2])n-[P1]- / CG2 CG1 / -([P2]-[P3]-[SSRS]-[P2])n- / CG2; in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid containing a gene of interest (GOI); and [SSRS] is a site-specific recombination site (SSRS). n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0019] In some aspects of the method disclosed above, the homologous recombination is mediated by CRISPR / Cas system, TALEN system or ZFN system. In some aspects, the CRISPR / Cas system further comprises single guide RNA (sgRNA). In some aspects of the method disclosed above, the site-specific recombinase recombination site (SSRS) is a Tyr recombinase site, a Tyr integrase site, a serine resolvase / invertase site or a serine integrase site. In some aspects, the Tyr recombinase site includes Cre, Dre, Flp, KD, B3 or B3 Tyr recombinase site. In some aspects, the Tyr integrase site includes λ (lambda), HK022 or HP1 Tyr integrase site. In some aspects, the serine resolvase / invertase site includes γδ (gamma delta), ParA, Tn3 or Gin serine resolvase / integrase site. In some aspects, the serine integrase site includes PhiC31, Bxb1, pr R4 serine integrase site. In some aspects, the Tyr recombinase site includes a Cre Tyr recombinase site. In some aspects, the SSRS is a LoxP site. In some aspects, the LoxP site includes the nucleic acid sequence shown in SEQ ID NO:1 (wild-type LoxP). In some aspects, the LoxP site includes a mutant LoxP site. In some aspects, the mutant LoxP site includes the nucleic acid sequence shown in SEQ ID NO:2 (mutant LoxP). In some aspects, the mutant LoxP site includes a nucleic acid selected from the following: SEQ ID NO:3 (Lox 511); SEQ ID NO:4 (Lox 5171); SEQ ID NO:5 (Lox 2272); SEQ ID NO:6 (Lox M2); SEQ ID NO:7 (LoxM3); SEQ ID NO:8 (Lox M7); SEQ ID NO:9 (Lox M11); SEQ ID NO:10 (Lox 71); and SEQ ID NO:11 (Lox 66). In some aspects, the mutant LoxP site includes any LoxP site disclosed in this specification. In some aspects, the Tyr recombinase site includes a Flp Tyr recombinase site. In some aspects, the SSRS is a short flip enzyme recognition target (FRT) site. In some aspects, the SSRS includes any FRT site sequence disclosed in this specification. In some aspects, the serine integrase site includes an att site, for example, attP or attB site. In some aspects, the SSRS includes any att site disclosed in this application.

[0020] In some aspects of the method disclosed above, the at least one selective marker and / or the at least one nucleic acid sequence encoding a detectable marker is glutamine synthetase (GS) and / or dihydrofolate reductase (DHFR). In some aspects, the at least one selective marker and / or the at least one nucleic acid sequence encoding a detectable marker is a drug resistance gene. In some aspects, the drug resistance gene is an antibiotic resistance gene. In some aspects, the antibiotic resistance gene is a puromycin resistance gene. In some aspects, the puromycin resistance gene is puromycin-N-acetyltransferase. In some aspects, the at least one selective marker and / or the at least one nucleic acid sequence encoding a detectable marker includes a protein. In some aspects, the protein is a fluorescent protein. In some aspects, the fluorescent protein is mCherry. In some aspects, the fluorescent protein includes GFP, ZsGreen1, AcGFP1, EGFP, GFPuv, AcGFP, EBFP, EYFP, ECFP, tdTomato, mCherry, DsRed, AmCyan, ZsGreen, ZsYellow, DsRed2, DsRed-Express, HcRed, AsRed, mOrange, mOrange2, mPlum, mStrawberry, mBanana, YFP, mRaspberry, HcRed1, E2-Crimson, or any combination thereof.

[0021] In some aspects of the method disclosed above, the cell is a Chinese hamster ovary (CHO) cell. In some aspects, the cell is HEK293 or NS0.

[0022] In some aspects of the methods disclosed above, the nucleic acid encoding the GOI encodes at least one polypeptide. In some aspects, the at least one polypeptide is an antibody or a fusion protein. In some aspects, the expression plasmid comprises one, two or more than two copies of the GOI, a detectable marker or a combination thereof.

[0023] In some aspects, the methods disclosed above further comprise determining the expression of the GOI, a detectable marker, or a combination thereof. In some aspects, the expression of the GOI is determined quantitatively and / or qualitatively. In some aspects, the expression of the GOI is determined by cell sorting, FACS, cell surface staining, Western blot, Northern blot, column chromatography, capillary electrophoresis, microfluidics, UV absorbance, immunohistochemistry, cell size, secreted protein levels, transcript levels, or any combination thereof.

[0024] In some aspects of the methods disclosed herein, the landing pad plasmid or the expression plasmid is integrated into the genome of the cell at a copy number of 1. In some aspects, the landing pad plasmid or the expression plasmid is integrated into the genome of the cell at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0025] In some aspects of the methods disclosed herein, (i) the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 18 or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 19 or a subsequence thereof; (ii) the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 114 or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 115 or a subsequence thereof; or (iii) the 5' homologous recombination site and the 3' homologous recombination site comprise polynucleotide sequences flanking the parent plasmid.

[0026] In some aspects of the methods disclosed herein, the parent plasmid comprises an open reading frame (ORF) encoding a first GOI, such as an antibody.

[0027] The present disclosure provides a landing pad cell comprising a plasmid having a topology corresponding to the following description: CG1 / -[P1]-[P2]-[SSRS]-[M]-[SSRS]-[P2]-[P1]- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-[P2]-[P1]- / CG2; CG1 / -[P1]- / CG2; CG1 / -([P1]-([P2])n-[P1])- / CG2; -[P1]-[P2]-[P1]-; or -([P1]-([P2])n-[P1])- in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; and [SSRS] is a site-specific recombination site (SSRS). n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0028] The present disclosure provides an expression cell comprising a plasmid having a topological structure corresponding to the following description: CG1 / -[P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; or CG1 / -([P2]-[P3]-[SSRS]-[P2])n- / CG2; in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid containing a gene of interest (GOI); and [SSRS] is a site-specific recombination site (SSRS). n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0029] The present disclosure provides a cell line produced by any of the methods disclosed herein. Also provided is a kit comprising the cells disclosed herein or cells produced according to any of the methods disclosed herein and instructions for their use.

[0030] The present disclosure also provides an isolated cell comprising a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI) integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or polynucleotide subsequence within SEQ ID NO:20 or SEQ ID NO:116.

[0031] Also provided is a method comprising introducing a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI) into a CHO cell and obtaining a CHO cell, wherein the nucleic acid is integrated at a specific locus of the genome of the CHO cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO: 116. Also provided is a method comprising providing a cell comprising a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), wherein the polynucleotide sequence comprises the nucleic acid encoding the gene of interest (GOI) operably linked to a promoter, wherein the nucleic acid is integrated at a specific locus of the genome of the CHO cell, and wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO: 116. In some aspects of the methods or isolated cells disclosed herein, (i) the nucleotide subsequence within SEQ ID NO: 20 comprises the sequence shown in SEQ ID NO: 21; or (ii) the nucleotide subsequence within SEQ ID NO: 116 comprises the sequence shown in SEQ ID NO: 117. In some aspects of the methods or isolated cells disclosed herein, (i) the nucleotide subsequence from within SEQ ID NO: 20 consists of the sequence shown in SEQ ID NO: 21; or (ii) the nucleotide subsequence from within SEQ ID NO: 116 consists of the sequence shown in SEQ ID NO: 117. In some aspects of the methods or isolated cells disclosed herein, (i) the nucleotide subsequence from within SEQ ID NO: 20 is a subsequence upstream (towards the 5' end of SEQ ID NO: 20) with respect to the sequence shown in SEQ ID NO: 21; or (ii) the nucleotide subsequence from within SEQ ID NO: 116 is a subsequence upstream (towards the 5' end of SEQ ID NO: 116) with respect to the sequence shown in SEQ ID NO: 117. In some aspects of the methods or isolated cells disclosed herein, (i) the nucleotide subsequence from SEQ ID NO:20 is a subsequence downstream (towards the 3' end of SEQ ID NO:20) of the sequence shown in SEQ ID NO:21; or (ii) the nucleotide subsequence from SEQ ID NO:116 is a subsequence downstream (towards the 3' end of SEQ ID NO:116) of the sequence shown in SEQ ID NO:117.

[0032] In some aspects, the methods, cells, cell lines or kits disclosed herein comprise the use of at least two landing pad plasmids or at least two expression plasmids. In some aspects, the two landing pad plasmids or the two expression plasmids are in a configuration selected from the following: head to head, tail to tail, tail to head and head to tail. In some aspects, each expression plasmid comprises at least a nucleic acid encoding a gene of interest (GOI). In some aspects, all GOIs are identical. In some aspects, all GOIs are different. In some aspects, at least one GOI is different from the rest. In some aspects, the first GOI comprises the heavy chain (HC) of an antibody, and the second GOI comprises the light chain (LC) of an antibody. In some aspects, at least one expression plasmid is bicistronic. In some aspects, the bicistronic expression plasmid encodes a first GOI comprising the HC of an antibody and a second GOI comprising the LC of an antibody. In some aspects, at least one landing pad plasmid is addressable. In some aspects, each landing pad plasmid comprises two Lox sites. In some aspects, the Lox sites are Lox P and Lox 511. In some aspects, each landing pad plasmid comprises a Lox site and a Frt site. In some aspects, each landing pad plasmid comprises one or two aat sites. In some aspects, each landing pad plasmid is addressable. In some aspects, each addressable landing pad plasmid comprises a pair of addressable SSRS unique to the landing pad. In some aspects, at least one pair of addressable SSRS is a pair of Lox sites. In some aspects, at least one pair of Lox sites is Lox 511 and Lox P. In some aspects, at least one pair of Lox sites is Lox m3 and Lox m7. In some aspects, a first addressable landing pad plasmid comprises Lox 511 and Lox P pairs of Lox sites, and a second addressable landing pad plasmid comprises Lox m3 and Lox m7 pairs of Lox sites. In some aspects, each addressable landing pad plasmid comprises an att site that is not cross-compatible.

[0033] The present disclosure also provides a cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO: 116 or an orthologous sequence thereof. Also provided is a cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 21 or SEQ ID NO: 117 or an orthologous sequence thereof. A cell is also provided, comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a target gene (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus of the genome of the cell, wherein the locus is a polynucleotide subsequence overlapping or covering SEQ ID NO: 20 or SEQ ID NO: 116 or a direct homologous sequence thereof. A cell is also provided, comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a target gene (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus of the genome of the cell, wherein the locus is a polynucleotide subsequence overlapping or covering SEQ ID NO: 21 or SEQ ID NO: 117 or a direct homologous sequence thereof. In some aspects, the cell is a CHO cell. In some aspects, the orthologous sequence has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 20, 21, 116, 117 or a subsequence thereof. In some aspects, sequence identity is determined via pairwise alignment using the implementation of the Needleman-Wunsch algorithm. In some aspects, the cell comprises two landing pad plasmids or two expression plasmids. In some aspects, the cell comprises more than two landing pad plasmids or more than two expression plasmids. In some aspects, both landing pad plasmids are addressable. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic diagram depicting a standard expression cell line development strategy, in which cells are transfected with an expression plasmid, resulting in its integration at a random location in the cell genome.

[0035] Figure 2Summarizes the strategy for identifying two parental cell lines suitable for landing pad cell development. Parental cell lines 1 and 2 are cell lines expressing monoclonal antibodies from parental plasmids for protein 1 and protein 2, respectively. Arrows and GS indicate glutamine synthetase complementation, mAb = monoclonal antibody; LC = light chain; HC = heavy chain; copy number = number of expression plasmids in the cell line (each expression plasmid contains LC and HC expression cassettes. The copy number is determined by qPCR using GAPDH as an internal control); spPCR = splinkeret PCR (a technique that allows identification of plasmid junction sequences). The levels of LC RNA and HC RNA are normalized to the levels found for the antibody against protein 1 (i.e., antibody against protein 1 = 1.00).

[0036] Figure 3 is a simplified depiction of the parental plasmid showing the configuration found in both cell line 1 and cell line 2. The parental plasmid in both cell lines is in a head-to-tail configuration. The configuration in cell line 1 and cell line 2 was determined by Southern blot analysis and determination of plasmid sequence junctions, where plasmid-plasmid fusions were detected. The arrow and GS indicate glutamine synthetase complementation.

[0037] Figure 4A and Figure 4B Two strategies for generating landing pad cell lines containing site-directed recombination sites (such as LoxP) are shown separately. In both strategies, the landing pad plasmid is introduced into the cell by homologous recombination, which is stimulated by restricting the genome of the parental cell line with a site-specific nuclease (e.g., CRISPR-associated nuclease (Cas)) represented by scissors. Figure 4A In the parental cell line, the parental cell line is identified based on its performance and the number of sites in its genome where the expression plasmid / cassette is present. Figure 4B In Figure 4A The parental cell line was used as the landing pad cell line. Figure 4A and Figure 4B In both, the sequence of the cell genome needs to be known, and the sequences homologous to the cell genome that are cloned to flank the expression cassette (homolog 1, homolog 2). mCherry indicates an open reading frame encoding a fluorescent marker, the LoxP site is a sequence used by the Cre recombinase, the arrow and GS indicate glutamine synthetase complementation, and the arrow and Puro indicate puromycin resistance.

[0038] Figure 5A and Figure 5B The general TI strategy of the present disclosure is schematically presented. The TI technology disclosed herein comprises in a parent cell line ( Figure 5A ) or in the landing pad cell line ( Figure 5B) using one or more site-specific endonucleases directed against the parental plasmid sequence to stimulate homologous recombination with the second DNA. Figure 5A ) can also serve as a landing pad cell line ( Figure 5B ). These strategies are in contrast to techniques that require knowledge and use of genome sequences (see, e.g., Figure 4A and Figure 4B ). The boxes with vertical and wavy lines next to the genomic sequence represent regions of homology between different plasmids. The solid boxes next to each homology region represent sequences targeted by CRISPR / Cas, which are present in Figure 5A The parental cell line or Figure 5B The scissors represent CRISPR / Cas, the mCherry open reading frame encodes a fluorescent marker, the LoxP site is the sequence used by the Cre recombinase, the arrow and GS represent glutamine synthetase complementation, and the arrow and Puro represent puromycin resistance.

[0039] Figure 5C Depicted is the sequence organization of an expression plasmid (P4) in an expression cell generated according to the methods disclosed herein. These figures show the location of sequences derived from the parental plasmid (P1), the landing pad plasmid (P2), and the second GOI plasmid (P3). "Cellular genome" refers to the flanking genomic sequences.

[0040] Figure 5D Shown is a general TI strategy using a single SSRS site. Here, site-specific endonucleases are directed to parental plasmid sequences in parental cell lines to stimulate homologous recombination. The frame with vertical and wavy lines next to the genomic sequence represents the homologous regions between different plasmids. The solid frame sequence next to each homologous region represents the sequence targeted by a sequence-specific endonuclease (e.g., CRISPR / Cas), which is present in the parental cell line, but not in the plasmid to be recombined into these cell lines. A single SSRS site is present at the landing pad cell line, and here attB is used as an example. Through a single SSRS site, a GOI plasmid (P3) will be inserted into the targeted locus. Scissors represent sequence-specific endonucleases (e.g., CRISPR / Cas), mCherry open reading frames encode exemplary fluorescent markers, and attB and attP sites are sequences used by integrase. Arrows represent promoters, and GS represents glutamine synthetase complementation. An is a poly A signal sequence. mAb is a monoclonal antibody expression cassette containing its own promoter and poly A signal.

[0041] Figure 5EThe TI strategy of using cell genome sequence for homologous recombination to produce landing pad is shown. Here, site-specific nuclease targets cell genome sequence to stimulate homologous recombination. The genomic sequence represents the homology region between the landing pad plasmid and the parent cell. The solid box next to each homology region represents the sequence targeted by CRISPR / Cas, which is present in the parent cell line but not in the plasmid to be recombined into these cell lines. A single SSRS site is present in the landing pad cell line, and attB is shown here as an example. Through a single SSRS recombination, the GOI plasmid (P3) will be inserted into the targeted locus. The scissors represent CRISPR / Cas, and the mCherry open reading frame encodes a fluorescent marker. The attB and attP sites are sequences used by integrase. Arrows represent promoters, and GS represents glutamine synthetase complementation. An is a poly A signal sequence. mAb represents a monoclonal antibody expression cassette containing its own promoter and poly A.

[0042] Fig. 5F Describes the basis Figure 5E 1 and 2. Sequence organization of an expression plasmid (P5) in an expression cell line generated using the methods described in , or using random integration into a new genomic locus. These figures show the location of sequences derived from the landing pad plasmid (P2) and the second GOI plasmid (P3). "Cellular genome" refers to the flanking genomic sequences. Since plasmid P1 was completely removed or not present in the locus, there is no P1 portion in this expression plasmid configuration.

[0043] Fig. 6A and Figure 6B The generation of landing pad cell lines according to the present disclosure is summarized. Fig. 6A Shown is the replacement of a monoclonal antibody (mAb) encoding plasmid in a parental cell line with a portion of a landing pad plasmid (e.g., a linear plasmid containing an open reading frame encoding mCherry and a puromycin resistance gene flanked by LoxP sites) to generate a landing pad cell line expressing a marker (e.g., mCherry). Fig. 6A A description of the components in can be found above in Figure 5A and Figure 5B in the description. Figure 6B The increased frequency of generating mCherry landing pad cell lines stimulated by the presence of the single guide RNA (sgRNA) required by CRISPR / Cas technology is shown. The percentage of clones with the desired phenotype of a single mCherry expression cassette and the absence of mAb (approximately 25%). Landing pad cell lines for TI are identified by their expression levels (mean fluorescence intensity MFI), transcript levels, and the stability of these two parameters during cell passage.

[0044] Fig. 7A and Figure 7B Shows the Figure 5A , Figure 5B , Fig. 6A and Figure 6B Practical application of targeted integration methodology in cells. Fig. 7A In the experiment, the mCherry expression cassette was exchanged with an expression cassette expressing an antibody against protein 3 (mAb 3) by using Cre recombinase. Single-cell cloning of cells expressing only mAb 3 was performed by Berkley Lights (BL) and FACS technology. The cells were expanded and Protein expression was assessed in a 250 bioreactor system and by 24 deep well fed-batch (24DW FB). Figure 7B The cell population obtained after complementation selection for GS was screened by FACS for cell surface expression of protein 3 (vertical axis) and expression of mCherry (horizontal axis). 5.24% of the cells expressed only protein 3, 90.06% expressed both proteins, 4.12% expressed only mCherry, and 0.68% did not express either protein. Cells with cell surface staining only with mAb against protein 3 (mAb3) are the desired cells. The productivity obtained from the screened clones is summarized in the text next to the FACS data.

[0045] Fig. 8A and Figure 8B The universal targeted integration (UTI) technology that can be implemented using four different strategies (Strategy A, Strategy B, Strategy C and Strategy D) is summarized. The universal TI technology disclosed herein involves the use of one or more site-specific nucleases directed against parental plasmid sequences in the parental cell line (not present in the landing pad plasmid) to stimulate homologous recombination with the landing pad plasmid. The advantage of this strategy is that it does not require knowledge of the flanking genomic DNA sequences. In the example Fig. 8A In the UTI technique depicted in , the parental expression plasmid in the parental cell line is replaced by a landing pad (Strategy A), or the parental expression plasmid is deleted and the landing pad is inserted into an alternative locus (locus) in the cell genome (Strategy B). In both cases, site-specific endonucleases are used to stimulate recombination. After the landing pad cell line is generated, it is used to prepare an expression cell line in which the landing pad is replaced with a second GOI using Cre recombinase. In the example of Figure 8BIn the UTI technology depicted in , a single SSRS site is used to generate expression cell lines in strategy C and strategy D. Boxes with vertical and wavy lines represent homology regions between different plasmids. Solid boxes represent sequences targeted, for example, by CRISPR / Cas, which are present in the parent expression plasmid but not in the plasmid to be recombined into these cell lines. Scissors represent CRISPR / Cas. The mCherry open reading frame encodes a fluorescent marker. LoxP and Lox511 sites are sequences used by Cre recombinase. attB and attP sites are sequences used by integrase. Arrows and GS encode GS complementation. Arrows and Puro encode puromycin resistance. The descriptions of these four alternative strategies are exemplary, and the components shown in the accompanying drawings (e.g., CRISPR / Cas, mCherry, Lox sites, att sites) can be replaced with functional equivalents disclosed in this specification.

[0046] Fig. 9 Shows the use Fig. 8A and Figure 8B Summary of data from landing pad cell lines prepared using the strategy described in . These figures show the increased frequency of generating mCherry landing pad cell lines stimulated by the presence of the single guide RNA (sgRNA) required for CRISPR / Cas technology. 25% of the clones had the desired phenotype containing the mCherry expression cassette and the absence of mAb from the parental cell line. Landing pad cell lines used for TI were identified by their mCherry gene copy number, expression level (mean fluorescence intensity MFI), transcript levels, and stability of these two parameters during cell passaging.

[0047] Fig.10 Summarizes the results of experiments using twelve landing pad cell lines to construct expressing cell lines using a second GOI plasmid encoding two copies of the mAb light chain and two copies of the mAb heavy chain and a plasmid encoding Cre. The percentage of expressing cell lines is the percentage of mCherry negative (red (-)) cells in the bulk culture after selection.

[0048] Fig.11A and Fig. 11B Results from experiments using five landing pad cell lines derived through cell line development are summarized. After single cell cloning, 32 expressing cell lines from each landing pad cell line were randomly selected, expanded and tested in a 24-deep well plate (DWP) 14-day fed-batch assay. This allowed for a comprehensive characterization of the potential of the landing pad cell lines. The data are summarized in Fig.11A in and Fig. 11B The data are presented in box-whisker plot.

[0049] Fig. 12AA head-to-head dual landing pad configuration is shown. Each landing pad contains two different SSRS sites for directed recombination. One GOI (mAb) is inserted into each landing pad locus by recombination. The resulting mAb expression plasmid is still in a head-to-head configuration.

[0050] Fig. 12B It is a depiction of the effect of the double landing pad configuration and the Cre recombinase on the double landing pad. One landing pad is shown as an arrow with a diagonal line, and the other is shown as an arrow with a braided pattern. Both landing pads use LoxP and Lox511. In the presence of Cre, the head-to-head and tail-to-tail configurations remain intact. In the other two configurations, one of the landing pads can be permanently deleted. The purpose of having two or more landing pads is, for example, to be able to prepare bispecific mAbs and increase titers. When under the control of the same regulatory sequence (e.g., the same promoter), the probability that multiple landing pads have the same activity is very high. In some aspects, multiple landing pads (e.g., 3, 4 or more) can be present in a 1:1 ratio or in an alternative ratio (e.g., 1:2 or 2:1).

[0051] Fig.13 The outcome of TI of the second GOI in head-to-head and tail-to-tail dual landing pad configurations is shown. One landing pad is shown as an arrow with a diagonal line and the other is shown as an arrow with a weave pattern. Both landing pads used LoxP and Lox511. The second GOI is shown as a solid rectangle. In both cases, the expressing cell line has two second GOIs.

[0052] Fig.14 The outcome of TI of a second GOI in tail-to-head and head-to-tail dual landing pad configurations is shown. One landing pad is shown as an arrow with a diagonal line and the other is shown as an arrow with a weave pattern. Both landing pads used LoxP and Lox511. The second GOI is shown as a solid rectangle. In both cases, two different expression cell lines were generated, one with one second GOI and the second with two second GOIs.

[0053] Fig.15 A dual landing pad configuration with Frt and Lox sites and a depiction of the effects of Cre and Flp recombinases on the dual landing pads are shown. One landing pad is shown as an arrow with a diagonal line and the other is shown as an arrow with a braided pattern. Both landing pads use LoxP and Frt. In the presence of Cre+Flp, the head-to-head and tail-to-tail configurations remain intact. In the other two configurations, one of the landing pads can be permanently deleted. This is equivalent to Fig. 12B Observed in.

[0054] Fig.16A depiction of a dual landing pad configuration using the same aatP sites flanking all landing pads and the fate after transfection of a second GOI plasmid and Int into cells is shown. One landing pad is shown as an arrow with a diagonal line and the other is shown as an arrow with a weave pattern. The GOI is shown as a solid rectangle.

[0055] Fig.17A and Fig. 17B Schematically presented is how a dual landing pad can be used to increase the diversity of expression of different subunits that are assembled to prepare a desired composite biological product. Solid and dashed arrows represent different components required to prepare a biological product. For illustrative purposes, a composite biological product requires at least one of each arrow. Each GOI plasmid can contain a different configuration of each composite biological product subunit (i.e., arrow) of a composite biological product. The second GOI can be composed of multiple arrows in different orders or each arrow itself. The second GOI plasmid is transfected into a dual landing pad cell line along with a recombinase. In order to change the level of subunit expression, different combinations of second GOI plasmids are transfected into the dual landing pad cell line. Shown are different transfections with the second GOI to obtain gene copy ratios of 1:2, 1:1, and 2:1 for solid and dashed arrows after TI is completed. For example, in Fig.17A In the 1:2 ratio, one second GOI contains one copy of the dashed arrow and the other second GOI plasmid contains both solid and dashed arrows in one of two configurations. Fig.17A As shown, this would require two separate transfections of the dual landing pad cell line. Clearly, this is not an exhaustive list of possible outcomes or inputs. Also not shown are configurations where a composite biologic is not made because only one subunit is incorporated into the landing pad (i.e., only one solid or only dashed arrow). Fig. 17B A simplified illustration of the use of addressable landing pads with unique SSRS is shown. One landing pad consists of Lox 511 and Lox P, and the second has Lox sites 2272 and M3. A second GOI plasmid will be specifically targeted to one landing pad or the other using the corresponding Lox site.

[0056] Fig.18 The utility of a dual landing pad with addressable landing pads is shown. One landing pad is shown as an arrow with diagonal lines and the other is shown as an arrow with a weave pattern. The second GOI plasmid is shown as a solid rectangle or a rectangle with vertical lines. In this example, each landing pad is flanked by a unique combination of Lox sites that only recombine with itself. This example is illustrative and other recombinases and their target sites can be used. Having addressable landing pads ensures that all four configurations of the dual landing pad have a defined second GOI without the need for multiple recombinases. Fig. 12B Shown missing one of the landing pads in the tail-to-nose and nose-to-tail configurations.

[0057] Fig.19 A diagram is shown demonstrating that having dual landing pads with a single aatB site in each landing pad eliminates landing pad deletions. One landing pad is shown as an arrow with a diagonal line and the other is shown as an arrow with a weave pattern. The second GOI plasmid is shown as a solid rectangle. Dual landing pads are addressable if the attP sites used are not cross-compatible.

[0058] Fig. 20 A proof of concept (POC) for targeted integration using a dual landing pad cell line is shown. The mCherry expression cassette is exchanged with the second GOI mAb expression cassette using Cre recombinase, as summarized in Figure 8b. The cell population obtained after GS complementation selection was screened by FACS for mCherry expression (horizontal axis) and cell surface expression of mAb (vertical axis). Here, 5.24% of the cells expressed only mAb, 90.06% expressed both proteins, 4.12% expressed only mCherry, and 0.68% did not express either of the two proteins.

[0059] Fig.21 Targeted integration of the GOI was shown to produce higher production cells compared to random integration. Secondary GOI plasmids, mAbs A and B, were integrated into the host cells by either random or targeted integration. The landing pad cell line is a direct descendant of the cell line used for random integration. The titer of the cell population used for single cell cloning was determined. The titer of the targeted integration population was approximately three to four times that of the random integration, demonstrating the value of this technology to generate landing pad cell lines that can outperform the industry standard of random integration.

[0060] Fig. 22 A summary of the preparation of expressing cell lines using a dual landing pad cell line with a second GOI plasmid containing either 1 LC + 1 HC or 2 LC + 2 HC is shown. Secondary GOI plasmids containing either 1 LC + 1 HC or 2 LC + 2 HC for mAbs A and B were used in TI cell line development. Productivity of the top 6 clones from each group is shown. In both cases, increasing the LC and HC copy number increased the mean titer by 25% to 37%, and the median titer by 35% to 37%. DETAILED DESCRIPTION

[0061] The present disclosure provides methods for generating landing pad cells, wherein a linear plasmid (e.g., a linear plasmid) comprising a gene of interest (e.g., one or more open reading frames encoding an antibody) can be inserted into the genome of a host cell without prior knowledge of the host cell genomic sequence for its targeted insertion. Although linear plasmids are generally preferred, circular plasmids can be used to generate landing pad cells.

[0062] The terms "targeted insertion" and "targeted integration" are used interchangeably to refer to a gene targeting method for directly inserting or integrating a gene or nucleic acid sequence into a specific position on a genome (i.e., directing a gene or nucleic acid sequence to a specific site between two nucleotides in a continuous polynucleotide chain). Targeted insertion can also be performed to introduce a small amount of nucleotides or to introduce an entire gene cassette, which comprises, for example, a plurality of genes, regulatory elements, and / or nucleic acid sequences. "Insertion" and "integration" and grammatical variants thereof are used interchangeably throughout the specification. In some aspects, targeted integration can be performed via recombination (e.g., site-specific recombination, homologous recombination, or a combination thereof).

[0063] According to these methods, cell lines (e.g., that have been historically known to exhibit favorable properties with respect to expression of a protein of interest (e.g., high recombinant protein yield, low protein degradation or misfolding, a particular glycosylation pattern, or other properties associated with post-translational modifications)) can be used as a parent cell line to generate a landing pad cell line that can be used to express other genes of interest. The parent cell line is ideally a cell that is hot (i.e., produces high titers of recombinant protein) and has one or more hotspots (genomic regions where introduction of foreign nucleic acid encoding a protein of interest will not be disruptive and will result in high levels of recombinant protein expression). As part of the parent cell selection process disclosed herein, two hotspots were identified.

[0064] A plasmid in a parent cell (parental plasmid) comprising an expression cassette integrated into the genome of a parental cell line is partially removed by excising (e.g., via homologous recombination) it between two locations (e.g., recombination sites) located in the interior of the parental plasmid (i.e., without cutting / destroying the parental cell genomic DNA), and replacing the excised region with another DNA sequence (landing pad plasmid) comprising two new recombination sites flanking at least one marker (e.g., a selectable and / or screenable marker). This method produces landing pad cells that can be used to insert a nucleic acid sequence (e.g., an expression plasmid or a gene of interest plasmid) comprising a different gene of interest (i.e., a gene of interest different from the gene of interest present in the parental cell) via recombination at the two newly introduced recombination sites. Different strategies related to this general process are disclosed in the present application.

[0065] These methods are generic in nature, allowing the use of any particularly advantageous parental cell line as a landing pad cell line based on knowledge of the parental plasmid sequence. Knowledge of the plasmid present in the parental cell line (parental plasmid), typically a commercial plasmid known in the art, readily allows the selection of recombination sites suitable for introduction of the landing pad plasmid, or a portion thereof, into the genome of the parental cell. In turn, the newly introduced recombination sites in the landing pad plasmid can be used to integrate a plasmid, or a portion thereof (e.g., a linear or circular plasmid), containing a gene of interest into the genome of the parental cell, thereby generating an expressing cell. See, e.g., Figure 4A , Figure 5A , Fig. 6A and Fig. 8A For example, in Fig. 8A (Strategy A and Strategy B) and Figure 8B (Strategy C and Strategy D) depicts a general targeted integration strategy. Also provided are constructs comprising multiple landing pads in different configurations (see, e.g., Fig. 12B ), where each pad can be created by using a unique SSRS combination (see, e.g., Fig.18 ) to uniquely identify.

[0066] Identification of a parental cell line as a "hot cell line" (i.e., having high production of recombinant protein or another favorable property) and subsequent identification of "hot spots" into which the parental plasmid is inserted supports methods for making and using landing pad cells, and improved expression of alternative related biological products (such as monoclonal antibodies) achieved using these methods and / or landing pad cells, without requiring knowledge of the sequence of the parental cell genome.

[0067] Therefore, the present disclosure also provides landing pad cells, landing pad plasmids, and kits containing, for example, reagents for generating landing pad cell lines and / or for generating expression cell lines. In addition to providing landing pad cells containing a single landing pad plasmid, the present disclosure also provides landing pad cells containing multiple landing pads. In some aspects, multiple landing pads in the landing pad cells of the present disclosure can be addressable, for example, by containing site-specific recombination sites or combinations thereof that uniquely identify each landing pad. I. Terminology

[0068] In order to more easily understand the present disclosure, some terms are first defined. As used herein, unless otherwise expressly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the present application.

[0069] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. The term "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably herein. In some aspects, the term "a" or "an" means "single". In other aspects, the term "a" or "an" includes "two or more" or "multiple".

[0070] Furthermore, where used herein, "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein with phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0071] The term "about" or "consisting essentially of..." refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of..." can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of..." can mean a range of up to 10%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude of a value or up to 5 times the value. When a specific value or composition is provided in the application and claims, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of..." is within an acceptable error range for that specific value or composition.

[0072] It will be understood that wherever aspects are described herein with the language "comprising," otherwise similar aspects described with the language "consisting of" and / or "consisting essentially of" are also provided.

[0073] As used herein, the term "approximately" as applied to one or more target values ​​refers to a value similar to the stated reference value. In certain aspects, unless otherwise stated or the context has a clear meaning, the term "approximately" refers to a series of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value (except where this number exceeds 100% of the possible value).

[0074] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the recited range and fractions of the recited value (such as tenths and hundredths of integers) where appropriate.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary of many terms used in the present disclosure for the skilled person.

[0076] Units, prefixes and symbols are expressed in their SI recognized form. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the defined terms are more fully defined by reference to the specification as a whole.

[0077] Abbreviations used herein are defined throughout this disclosure. Various aspects of this disclosure are described in further detail in the following subsections.

[0078] Nucleotides are represented by their generally accepted single-letter codes. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction. Nucleotides are represented herein by the commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0079] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation.

[0080] The terms "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified forms of polynucleotides, such as by alkylation and / or by capping, and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) including mRNA and gRNA (whether spliced ​​or unspliced), any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, and other polymers containing a normucleotidic backbone (e.g., polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that permits base pairing and base stacking (as found in DNA and RNA).

[0081] The terms "nucleic acid sequence" and "nucleotide sequence" are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be single-stranded or double-stranded DNA or RNA (e.g., gRNA).

[0082] As used herein, the term "subsequence" refers to a subset of contiguous nucleotides in a sequence (either a physical sequence or a symbolic representation thereof).

[0083] The methods disclosed herein can be used, for example, to produce biologics (eg, antibodies).

[0084] As used herein, the term "antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin (which specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds) or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region contains three constant domains, namely C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region contains a constant domain: C L . VH and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It comprises three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Thus, for example, the term "anti-PD-1 antibody" includes whole antibodies that specifically bind to PD-1, have two heavy chains and two light chains, and antigen-binding portions of whole antibodies. Non-limiting examples of antigen-binding portions are shown elsewhere herein. In some aspects of the present disclosure, the anti-PD-1 antibody is nivolumab or an antigen-binding portion thereof.

[0085] In some aspects, the antibody is a bispecific antibody. "Bispecific antibody" is a specific type of "bispecific molecule" or "bispecific binding molecule". The term "bispecific antibody" means an antibody that can be combined with at least two antigenic determinants (e.g., epitopes) through two different antigen binding sites. In some aspects, a bispecific antibody can combine two antigenic determinants (e.g., epitopes) in parallel. In some aspects, a bispecific antibody combines a kind of antigen (or epitope) on one of its binding arms (a pair of heavy chains / light chains), and combines different antigens (or epitopes) on its second binding arm (a different pair of heavy chains / light chains). In some aspects, a bispecific antibody can have two different antigen binding arms (both specificity and CDR sequences are different), and is monovalent for each antigen combined with it. Bispecific antibodies include, for example, those produced by quadroma technology (Milstein & Cuello (1983) Nature 305(5934):537-40), chemical conjugation of two different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012):628-31), or knob-in-hole or similar methods that introduce mutations in the Fc region (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):6444-6448).

[0086] In the recent past, a variety of recombinant antibody formats (e.g., trivalent or tetravalent bispecific antibodies) have been developed. Examples include fusions of IgG antibody formats and single chain domains (for different formats, see, e.g., Coloma, MJ et al., Nature Biotech 15 (1997), 159-163; WO 2001 / 077342; Morrison, SL, Nature Biotech 25 (2007), 1233-1234; Holliger. P. et al., Nature Biotech. 23 (2005), 1 126-1136; Fischer, N. and Leger, O., Pathobiology 74 (2007), 3-14; Shen, J. et al., J. Immunol. Methods 318 (2007), 65-74; Wu, C et al., Nature Biotech. 25 (2007), 1290-1297). Bispecific antibodies include trivalent or tetravalent bispecific antibodies produced according to the methods disclosed in WO 2009 / 080251; WO 2009 / 080252; WO 2009 / 080253; WO 2009 / 080254; WO 2010 / 112193; WO 2010 / 115589; WO 2010 / 136172; WO 2010 / 145792; WO 2010 / 145793 and WO 2011 / 117330, all of which are incorporated herein by reference in their entirety. One of ordinary skill in the art will appreciate that higher order valencies may also be used.

[0087] In the recent past, various recombinant bispecific antibody formats have been developed, for example by fusion of IgG antibody formats and single chain domains (see Kontermann RE, mAbs 4:2, (2012) 1-16). Bispecific antibodies in which the variable domains VL and VH or the constant domains CL and CH1 are replaced by one another are described in WO 2009080251 and WO2009080252.

[0088] The method to circumvent the problem of mispaired byproducts, which is called "knob-to-hole structure", aims to force the pairing of two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. On one chain, bulky amino acids are replaced by amino acids with short side chains to produce a "hole". In contrast, amino acids with large side chains are introduced into the other CH3 domain to produce a "knob". By co-expressing the two heavy chains (and two identical light chains, which must fit into the two heavy chains), the formation of heterodimers ("knob-to-hole") was observed in high yields compared to the formation of homodimers ("hole-to-hole" or "knob-to-hole") (Ridgway JB, Presta LG, Carter P; and WO 1996027011). The percentage of heterodimers can be further increased by using phage display methods to reshape the interaction surfaces of the two CH3 domains and introduce disulfide bonds to stabilize heterodimers (Merchant AM et al., Nature Biotech 16 (1998) 677-681; Atwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). New methods for knob-in-hole technology are described in, for example, EP 1870459A1. Xie, Z., et al., J Immunol Methods 286 (2005) 95-101 refer to the format of bispecific antibodies using scFv combined with knob-in-hole technology for the Fc portion. See, Godar et al. (2018) “Therapeutic bispecific antibody formats: a patent applications review (1994-2017)” Expert. Opin. Ther. Pat. 28(3): 251-276, and Brinkmann and Kontermann (2017) “The making of bispecific antibodies” mAbs 9: 182-212; both of which are incorporated herein by reference in their entirety.See also, Ridgway et al. (1996) Protein Eng 9:617-21; Atwell et al. (1997) J. Mol. Biol. 270:26-35; Merchant et al. (1998) Nat. Biotechnol. 16:677-681; Moore et al. (2011) MAbs 3:546-55; Von Kreudenstein et al. (2013) MAbs 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem. 285:19637-47; Geuijen et al. (2014) J. Clin. Oncology 32: Suppl:560; Strop et al. (2012) J. Mol. Biol. 420:204-19; Choi et al. (2013) Mol. Cancer Ther. 12:2748-59; Choi et al. (2015) Mol. Immunol. 65:377-83; Labrijn et al. (2013) Proc. Natl. Acad. Sci. USA 110:5145-50; Davis et al. (2010) Protein Eng. 23:195-202; Moretti et al. (2013) BMC Proceedings 7(Suppl 6):09; and Leaver-Fey et al. (2016) Structure 24:641-51, all of which are incorporated herein by reference in their entirety. Light chain pairing strategies are disclosed, for example, in Schaefer et al. (2011) Proc Natl Acad Sci US A. 108(27): 11187-92; Lewis et al. (2014) Nat Biotechnol. 32(2): 191-8; Mazor et al. (2015) MAbs. 7(2): 377-89; Liu et al. (2015) J Biol Chem. 290(12): 7535-62; Dillon et al. (2017) MAbs. 9(2): 213-230; and U.S. Pat. No. 9,914,785, all of which are incorporated herein by reference in their entirety.

[0089] Immunoglobulin can be derived from any well-known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art, and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in the human body. In the absence of explicit instructions, unless otherwise indicated by the context, the term "antibody" also includes any of the above-mentioned antigen-binding fragments or antigen-binding portions of the immunoglobulin, and includes monovalent and divalent fragments or portions and single-chain antibodies.

[0090] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to an antigen (e.g., PD-1) is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity with other antigens (e.g., PD-1 molecules from different species). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0091] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules having a single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0092] "HuMAb" refers to an antibody with a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0093] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulin. In one aspect of the humanized form of the antibody, some, most or all of the amino acids outside the CDR have been replaced by amino acids from human immunoglobulin, while some, most or all of the amino acids in one or more CDRs are unchanged. A small amount of addition, deletion, insertion, substitution or modification of amino acids is permissible as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized antibody" retains an antigenic specificity similar to the original antibody.

[0094] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0095] "Anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to the PD-1 antigen, and an anti-PD-L1 antibody specifically binds to the PD-L1 antigen.

[0096] An "antigen-binding portion" of an antibody (also referred to as an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the intact antibody. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) include (i) a Fab fragment (fragment from papain cleavage) or a V L 、V H (ii) a F(ab')2 fragment (fragment derived from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a fragment consisting of V H and CH1 domains; (iv) a V fragment consisting of a single arm of the antibody L and V H (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which are composed of V H (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs that may be optionally connected by a synthetic linker. In addition, although the two domains V of the Fv fragment L and V H are encoded by separate genes, but they can be joined by synthetic linkers using recombinant methods to make them into a single protein chain in which V L and V HThe antibody fragments are obtained by pairing the fragments with the corresponding regions to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0097] In some respects, biological products can be proteins, polypeptides or polynucleotides. In some respects, biological products are enzymes, receptors, receptor ligands, protein antibiotics, fusion proteins, structural proteins, regulatory proteins, vaccines, growth factors, hormones or cytokines. In some respects, biological products can include one or more heterologous parts, such as parts that extend the plasma half-life of biological products, parts that promote transmembrane or trans-blood-brain barrier transport, parts that increase or reduce clearance, or parts (that is, targeting parts) that guide biological products to specific cells or tissue types.

[0098] An "isolated" polynucleotide, vector, polypeptide, cell or any composition disclosed herein is a polynucleotide, vector, polypeptide, cell or composition that is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some aspects, an isolated polynucleotide, vector, polypeptide or composition is substantially pure.

[0099] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; the modification is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids such as homocysteine, ornithine, para-acetylphenylalanine, D-amino acids and creatine) as well as other modifications known in the art.

[0100] The term "percentage of sequence identity" between two polypeptide or polynucleotide sequences refers to the number of identical matching positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., spaces) that must be introduced to achieve optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a space is not a nucleotide or amino acid, the space present in the target sequence is not counted. Similarly, since the target sequence nucleotides or amino acids are counted instead of the nucleotides or amino acids in the reference sequence, the space present in the reference sequence is not counted. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent.

[0101] The sequence identity percentage is calculated in the following manner: determine the number of positions where the same amino acid residue or nucleic acid base occurs in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using readily available software (for online use and for downloading). Suitable software programs can be obtained from various sources and can be used to compare protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. Use BLASTN to compare nucleic acid sequences, and use BLASTP to compare amino acid sequences. Other suitable programs are for example Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) website www.ebi.ac.uk / Tools / psa.

[0102] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that the percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0103] In certain aspects, the percent identity "ID%" of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as ID%=100x(Y / Z), wherein Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences (such as by visual inspection or alignment with a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than that of the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0104] Those skilled in the art will appreciate that the generation of sequence alignments for calculating percentages of sequence identity is not limited to binary sequence-sequence comparisons driven only by raw sequence data. It should also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structure), functional data (e.g., position of mutations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available from www.tcoffee.org, or alternatively, for example, from EBI. It should also be understood that the final alignment for calculating percentages of sequence identity can be automatically or manually compiled.

[0105] The terms "gene", "coding sequence", "coding nucleic acid", "open reading frame", "ORF" and grammatical variants thereof are used interchangeably in this disclosure and refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a gene of interest (GOI), which is typically a protein (e.g., a biological product such as an antibody). The coding sequence may further comprise start and stop signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0106] As used herein, the term "gene of interest" (abbreviated as "GOI") refers to an exogenous protein to be expressed by a cell disclosed herein. In some aspects, a GOI is a biological product, such as an antibody or a portion thereof. In some aspects, a GOI comprises one or more open reading frames, such as encoding one or more recombinant proteins, operably linked to one or more promoters and / or other regulatory sequences. In some aspects, a cell disclosed herein may contain a first GOI, which may be replaced by a second GOI. In some aspects, a first GOI (e.g., a GOI located on a parent plasmid) and a second GOI (e.g., a GOI located on a second GOI plasmid) belong to the same molecular class. For example, if the first GOI is an antibody, the second GOI may also be an antibody because the parent cell line effectively expresses this type of recombinant protein. In some aspects, a GOI is a nucleic acid (e.g., a therapeutic nucleic acid). In some aspects, the terms GOI and ORF may be used interchangeably, particularly when the GOI is encoded by a single ORF. In some aspects, a GOI may be encoded by more than one ORF. In some aspects, a GOI may be a detectable molecule (e.g., a marker).

[0107] As used herein, "complementary" or "complementary" refers to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. "Complementarity" refers to a property shared between two nucleic acid sequences such that when they are arranged antiparallel to each other, the nucleotide bases at each position will be complementary.

[0108] The terms "vector", "expression vector", "plasmid" and grammatical variants thereof are used interchangeably in this disclosure and refer to a polynucleotide that is foreign to the genome of a host cell and is inserted into a specific location in the genome of the host cell. Typically, a plasmid comprises multiple elements such as recombination sites (e.g., homologous recombination sites and / or site-specific recombination sites), markers (e.g., detection markers and / or selection markers), one or more expression cassettes, or any combination thereof. In some aspects, a plasmid can be a linear plasmid. In other aspects, a plasmid can be a circular plasmid (e.g., a complete circular plasmid).

[0109] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship that permits them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0110] As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell), or a cell from a multicellular organism cultured as a unicellular entity (e.g., a cell line) in vivo or in vitro that can or has been used as a recipient of a heterologous nucleic acid, thus becoming a recombinant host cell. Thus, the term host cell also includes the progeny (i.e., recombinant host cells) of the original host cell (i.e., the host cell before receiving the heterologous nucleic acid) that has been transformed with a heterologous nucleic acid. It should be understood that the morphology or genome or total DNA complement of the progeny of a single cell may not necessarily be exactly the same as the original parent due to natural, accidental or deliberate mutations.

[0111] A "recombinant host cell" or "genetically modified host cell" is a host cell into which a heterologous nucleic acid (e.g., an expression vector) has been introduced. For example, a eukaryotic host cell becomes a recombinant or genetically modified eukaryotic host cell (e.g., a mammalian host cell) by introducing an exogenous nucleic acid into the eukaryotic host cell.

[0112] As used herein, the terms "hot cells," "hot clones," and "hot cell lines" refer to cells, clones, or cell lines, respectively, that have favorable properties, such as high yields of recombinant proteins compared to other cells, clones, or cell lines expressing the same recombinant protein. For example, hot cells, hot clones, or hot cell lines can express higher amounts of recombinant proteins, can express higher levels of correctly folded recombinant proteins, can express recombinant proteins with lower levels of high molecular weight aggregation, can express recombinant proteins with lower levels of fragmentation, or any combination thereof or some other desired property.

[0113] As used herein, the term "hotspot" refers to such a genomic position (locus) in which an exogenous sequence (e.g., a plasmid comprising a polynucleotide sequence encoding a protein for recombinant expression) can be inserted, and wherein (i) the transcription of the exogenous sequence is not silenced (e.g., by epigenetic modification) and (ii) the transcription of the exogenous sequence occurs at a high level compared to the transcription level observed when the exogenous sequence is inserted at other positions (e.g., a reference position). In some aspects, the hotspot does not contain a functional ORF. Therefore, in some aspects, the hotspot does not contain one or more active transcription genes. Hotspots lacking active transcription genes are particularly advantageous because they are partially or completely deleted to insert a polynucleotide sequence encoding an exogenous gene (target gene) without destroying the production of endogenous proteins. In some aspects, the hotspot of the present disclosure is located near an active transcription gene or between two active transcription genes, i.e., the hotspot can be flanked by two active transcription genes. In some aspects, inserting a polynucleotide sequence encoding an exogenous gene (target gene) in a hotspot of the present disclosure does not affect the expression of one or more active transcription genes adjacent to the hotspot or flanking the hotspot. In some aspects, insertion of a polynucleotide sequence encoding an exogenous gene (gene of interest) in a hotspot of the present disclosure reduces the expression of one or more actively transcribed genes adjacent to or flanking the hotspot by less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than about 10%.

[0114] As used herein, the term "addressable" applied to the polynucleotide sequences disclosed herein (e.g., landing pad sequences disclosed herein) refers to polynucleotide sequences uniquely identified by the presence of a unique site-specific recombination site (SSRS) or a combination thereof. For example, a first landing pad having a Lox 511 and lox P site and a second landing pad having a Lox m3 and Lox m7 site will be addressable with respect to each other. Therefore, in some aspects, the landing pad may be addressable due to the presence of a specific combination of two SSRS. In other aspects, the landing pad may be addressed with respect to the second landing pad via a single SSRS; for example, the first landing pad may have a first single aatP site, and the second landing pad may have a second single aatP site, wherein the aatP site is not cross-compatible. In some aspects of the present disclosure, there may be a plurality of connected landing pads, wherein each landing pad is uniquely addressable due to the presence of a unique SSRS or a combination thereof that specifically identifies (addresses) a given landing pad.

[0115] As used herein, the term "addressable SSRS" refers to a unique SSRS or combination thereof that can be specifically targeted for recombination. As used herein, the term "addressable landing pad plasmid" refers to a landing pad plasmid comprising an addressable SSRS or combination thereof that can be specifically targeted for recombination.

[0116] As used herein, the term "non-cross-compatible" when applied to a pair of site-specific recombination sites refers to sites that are defective in recombination with alternative SSRS (i.e., cannot recombine with alternative SSRS or have only some residual cross-reactivity). For example, two Lox sites (such as LoxP and Lox511) with reduced recombination potential with each other would be considered non-cross-compatible. Similarly, two attP-aatB site pairs with reduced recombination potential with each other would be considered non-cross-compatible.

[0117] As used herein, the terms "head to head", "tail to tail", "tail to head", and "head to tail" refer to the relative orientation of two polynucleotide sequences (e.g., two landing pads, landing pad plasmids, expression plasmids, or target genes in a genetic construct disclosed herein). The term "head" refers to the 5' end of a nucleic acid sequence and the term "tail" refers to the 3' end of a nucleic acid sequence. Thus, a 3'-5'5'-3' configuration is head to head because (considering the 5' to 3' end of the construct) the two 5' ends (heads) of the original sequence are adjacent to each other. A 5'-3'3'-5' configuration would therefore be tail to tail, 5'-3'5'-3' would be tail to head, and 3'-5'3'-5' would be head to tail. II. Landing Pad Cells

[0118] The present disclosure provides landing pad cells that can be used for the recombinant expression of at least one gene of interest (GOI). In some aspects, these cell lines contain a "landing pad" (i.e., one or more specific polynucleotide sequences inserted into the genome of a parent cell) that can be replaced, for example, via recombination, by another one or more specific polynucleotide sequences comprising a nucleotide sequence encoding at least one GOI. In some aspects, instead of replacing a polynucleotide sequence, for example, via recombination, one or more specific polynucleotide sequences comprising a nucleotide sequence encoding at least one GOI can be inserted at a position within the landing pad, for example, via an aat site.

[0119] As a general description of one of the processes disclosed herein, a parental cell line (e.g., a "historical" cell line known to efficiently express a particular biologic) is modified by fully or partially replacing an exogenous polynucleotide sequence (i.e., a "parent plasmid") comprising a parent or first GOI with a second exogenous polynucleotide sequence (i.e., a "landing pad plasmid or portion thereof"). The resulting cell line incorporating the landing pad plasmid or portion thereof (but not the entire parent plasmid) will be a "landing pad cell". In some aspects, a landing pad plasmid of the present disclosure comprises flanking sequences from the parent plasmid.

[0120] In turn, the landing pad plasmid in the landing pad cell can be replaced (e.g., in part) via recombination with another polynucleotide ("GOI plasmid") comprising a different or second GOI, thereby generating an "expressing cell." See, e.g., Figure 5A and Figure 5B and the process depicted in Table 1 . Table 1: Elements and recombination events *In some aspects, the parent plasmid is referred to as the "first GOI plasmid".

[0121] Thus, in some aspects, the disclosure provides expression cells comprising at least one expression plasmid (P4) (e.g., a linear plasmid) integrated into a genomic sequence, wherein each expression plasmid comprises (i) a polynucleotide sequence derived from an expression plasmid (P4), which comprises a nucleic acid encoding a gene of interest (second GOI); (ii) two SSRS (e.g., if a recombinase system such as Lox is used) or a single SSRS (e.g., if an integrase system such as att is used) flanking the polynucleotide sequence of (i); (iii) a polynucleotide sequence distal to the polynucleotide of (i) and the SSRS of (ii), wherein both flanking polynucleotide sequences of (iii) are derived from a landing pad plasmid (P2); (iv) a polynucleotide sequence flanking distally of the polynucleotide sequence of (iii), wherein both flanking polynucleotide sequences of (iv) are derived from the parent plasmid (P1).

[0122] As used herein, the term "site-specific recombination site" (abbreviated as "SSRS") includes a nucleotide sequence that can be recognized by a site-specific recombinase and acts as a substrate for a recombination event. In some aspects, the constructs disclosed herein (e.g., landing pad plasmids or expression plasmids) may include two SSRS, one located upstream and one located downstream of the nucleic acid encoding the GOI or marker. In some aspects, the constructs disclosed herein (e.g., landing pad plasmids or expression plasmids) may include a single SSRS located upstream or downstream of the nucleic acid encoding the GOI or marker. In some aspects, the constructs disclosed herein (e.g., landing pad plasmids or expression plasmids) may include more than two SSRS, all of which are located upstream of the nucleic acid encoding the GOI or marker, all of which are located downstream of the nucleic acid encoding the GOI or marker, or some of which are located upstream of the nucleic acid encoding the GOI or marker and some of which are located downstream thereof.

[0123] In the formulas disclosed in the present application that contain two SSRS, it is understood that if a system that requires a single SSRS (e.g., att) is used for recombination instead of a recombination system that requires two SSRS (e.g., lox or Frt), then one of the two SSRS in the formula is optional and may not be present. When one of the SSRS sites in the above formula is absent, the single SSRS site may be an SSRS upstream or downstream of the [M] or [P3] component. In some aspects, the single SSRS is an att site.

[0124] As used herein, the term "site-specific recombinase" includes a group of enzymes that can achieve the recombination between "recombination sites", wherein two recombination sites are located within a single nucleic acid molecule or on a separate nucleic acid molecule. The example of a "site-specific recombinase" includes but is not limited to Cre, Flp and Dre recombinases. In some aspects, the site-specific recombinase is an integrase, for example, λ (lambda) integrase. In some aspects, the site-specific recombinase is a Bxb integrase, for example, Bxb1 integrase. Bxb1 (integrase encoded by mycobacteriophage Bxb1) is a member of the serine recombinase family and catalyzes the chain exchange between attP and attB (attachment site of phage and bacterial host) respectively.

[0125] The present disclosure provides landing pad cells comprising at least one plasmid (e.g., a linear plasmid or a circular plasmid or a combination thereof) integrated into their genomic sequence, wherein each plasmid comprises (i) a polynucleotide sequence derived from a landing pad plasmid (P2) comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker (if a recombinase system such as Lox is used) or a single SSRS (if an integrase system such as att is used); and (ii) polynucleotide sequences flanking the polynucleotide sequence of (i), wherein both flanking polynucleotide sequences of (ii) are derived from the parent plasmid (P1).

[0126] It will be understood that where a single SSRS is present, describing its position as "flanking" another element in a formula, e.g., a [P2] or [P3] element (i.e., an element encoding a marker or GOI) refers to the SSRS's immediate upstream or downstream position with respect to the flanked element. As an example, the [SSRS] in the formula CG1 / -[P1]-[P2]-[SSRS]-[P3]-[P2]-[P1]- / CG2 would flank [P3] (which would encode a GOI).

[0127] The present disclosure provides an expression cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following formula CG1 / -[P1]-[P2]-[SSRS]-[P3]-[SSRS]-[P2]-[P1]- / CG2 in CG1 and CG2 are parental cell genomic sequences flanking the inserted linear plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a second GOI plasmid comprising a gene of interest (GOI); and [SSRS] is site-specific recombination site (SSRS).

[0128] The present disclosure also contemplates landing pad cells comprising multiple plasmids (e.g., landing plasmids or portions thereof). Thus, the present disclosure also provides landing pad cells comprising at least one plasmid (e.g., one, two, three or more linear plasmids or circular plasmids) integrated into its genomic sequence, wherein each plasmid comprises: a polynucleotide sequence derived from a landing pad plasmid (P2), the polynucleotide sequence comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker; and polynucleotide sequences flanking the polynucleotide sequence of (i), wherein both flanking polynucleotide sequences of (ii) are derived from a parent plasmid (P1).

[0129] Thus, the present disclosure provides an expression cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following formula CG1 / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG2 in CG1 and CG2 are parental cell genomic sequences flanking the inserted linear plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid containing a gene of interest (GOI); [SSRS] is a site-specific recombination site (SSRS); and n is an integer between 1 and 10.

[0130] In some aspects, [P3] can comprise a single GOI or multiple GOIs. In some aspects, 5' [SSRS] or 3' [SSRSA] is optional. In some aspects, the expression cell comprises a plasmid, wherein the plasmid is an expression plasmid.

[0131] In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, all plasmids are the same. In some aspects, all plasmids are different. In some aspects, at least one plasmid is different.

[0132] In some aspects, CG1 comprises the polynucleotide sequence of SEQ ID NO: 18 or a fragment thereof. In some aspects, CG2 comprises the polynucleotide sequence of SEQ ID NO: 19 or a fragment thereof.

[0133] In some aspects, CG1 comprises the polynucleotide sequence of SEQ ID NO: 114 or a fragment thereof. In some aspects, CG2 comprises the polynucleotide sequence of SEQ ID NO: 115 or a fragment thereof.

[0134] The present disclosure provides a landing pad cell comprising at least one plasmid (e.g., a linear plasmid) integrated into its genomic sequence, wherein the plasmid comprises a. a polynucleotide sequence comprising a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; b. two SSRS flanking the polynucleotide sequence of (1); and c. Two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2), which are homologous to the corresponding homologous recombination sites in the parental plasmid.

[0135] In some aspects, the topology of a plasmid (e.g., a linear plasmid) in a landing pad cell corresponds to the following formula CG1 / -[P1]-[P2]-[SSRS]-[M]-[SSRS]-[P2]-[P1]- / CG2 in CG1 and CG2 are parental cell genomic sequences flanking the inserted linear plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one marker (e.g., a screenable marker, a selectable marker, or a combination thereof); and [SSRS] is site-specific recombination site (SSRS).

[0136] In some aspects, the topology of a plasmid (e.g., a linear plasmid) in a landing pad cell corresponds to the following formula CG1 / -([P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1])- / CG2 in CG1 and CG2 are parental cell genomic sequences flanking the inserted linear plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one marker (e.g., a screenable marker, a selectable marker, or a combination thereof); [SSRS] is a site-specific recombination site (SSRS); and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, all plasmids are the same. In some aspects, all plasmids are different. In some aspects, at least one plasmid is different.

[0137] The present disclosure also provides a landing pad cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following description CG1 / -[P1]-[P2]-[P1]- / CG2 in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from a landing pad plasmid, comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker.

[0138] The present disclosure also provides a landing pad cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following description CG1 / -[P1*]- / CG2 CG3 / -[P2]- / CG4 in CG1 and CG2 are parental cell genomic sequences flanking the plasmid inserted in the first hotspot; CG3 and CG4 are parental cell genomic sequences flanking the plasmid inserted in the second hotspot; [P1*] is a polynucleotide sequence derived from a parent plasmid having at least a partial deletion; [P2] is a polynucleotide sequence derived from a landing pad plasmid, comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker.

[0139] The present disclosure also provides a landing pad cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following description CG1 / -([P1]-([P2])n-[P1])- / CG2 in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from a landing pad plasmid, the polynucleotide sequence comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker; and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, all plasmids are the same. In some aspects, all plasmids are different. In some aspects, at least one plasmid is different.

[0140] The present disclosure also provides a landing pad cell comprising a plasmid (e.g., a linear plasmid) inserted into its genomic sequence, wherein the topology of the plasmid corresponds to the following description CG1 / -([P1*]- / CG2 CG3 / -([P2])n- / CG4 in CG1 and CG2 are parental cell genomic sequences flanking the plasmid inserted in the first hotspot; CG3 and CG4 are parental cell genomic sequences flanking the plasmid inserted in the second hotspot; [P1*] is a polynucleotide sequence derived from a parent plasmid having at least a partial deletion; [P2] is a polynucleotide sequence derived from a landing pad plasmid, the polynucleotide sequence comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker; and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, all plasmids are the same. In some aspects, all plasmids are different. In some aspects, at least one plasmid is different.

[0141] In some aspects, CG1 comprises the polynucleotide sequence of SEQ ID NO: 18 or a fragment thereof. In some aspects, CG2 comprises the polynucleotide sequence of SEQ ID NO: 19 or a fragment thereof.

[0142] In some aspects, CG3 comprises the polynucleotide sequence of SEQ ID NO: 114 or a fragment thereof. In some aspects, CG4 comprises the polynucleotide sequence of SEQ ID NO: 115 or a fragment thereof.

[0143] In some aspects, for example, when a linear plasmid is inserted into a hotspot that is different from the original hotspot in the parental cell line, the CG1 and CG2 genomic sequences (the parental cell genomic sequences flanking the inserted linear plasmid) will be replaced by the CG3 and CG4 genomic sequences corresponding to the genomic sequences flanking the inserted linear plasmid in the alternative hotspot, respectively.

[0144] The present disclosure also provides a landing pad plasmid for targeted integration into a host cell genome comprising a plasmid (e.g., a linear plasmid), wherein the topology of the plasmid corresponds to the following formula -[P1]-[P2]-[P1]- in [P1] is a polynucleotide sequence derived from a parent plasmid integrated into the host comprising a homologous recombination site; and [P2] is a polynucleotide sequence comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker.

[0145] The present disclosure also provides a landing pad plasmid for targeted integration into a host cell genome comprising a plasmid (e.g., a linear plasmid), wherein the topology of the plasmid corresponds to the following formula -([P1]-([P2])n-[P1])- in [P1] is a polynucleotide sequence derived from a parent plasmid integrated in a host comprising a homologous recombination site; [P2] is a polynucleotide sequence comprising at least one marker and two site-specific recombination sites (SSRS) flanking the at least one marker; and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, all plasmids are the same. In some aspects, all plasmids are different. In some aspects, at least one plasmid is different.

[0146] It will be appreciated that the abbreviated topologies of the plasmids disclosed herein (eg, -[P1]-[P2]-[P1]-) may be described interchangeably using the terms "description" or "formula."

[0147] The present disclosure also provides a method of generating landing pad cells, the method comprising: (a) integrating the landing pad plasmid or a portion thereof into the genome of a parent cell at a targeted integration site using homologous recombination, The landing pad plasmid contains (1) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2) two SSRS flanking the polynucleotide sequence of (1); and (3) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2), which are homologous to the corresponding homologous recombination sites in the parental plasmid; and The homologous recombination sites of the landing pad plasmid recombine with the corresponding homologous recombination sites of the parental plasmid, thereby integrating the landing pad plasmid at an internal position within the parental plasmid inserted into the genomic DNA of the parental cell.

[0148] The targeted integration process disclosed herein involves replacing a polynucleotide subsequence located between two recombination sites in a plasmid with another polynucleotide subsequence located between two corresponding recombination sites in another plasmid. Figure 4A , Figure 5A , Fig. 8A and Figure 8B As exemplified in , targeted integration of a landing pad plasmid into a parental plasmid replaces a subsequence of the parental plasmid with a corresponding subsequence from the landing pad, retaining a remnant of the sequence from the parental plasmid between the recombination site and the genomic sequence. This targeted integration does not require a complete replacement of the plasmid by another plasmid.

[0149] Similarly, when the second GOI plasmid recombines with the landing pad plasmid, subsequences between the SSRS (e.g., LoxP sites) will be exchanged, but the remnant of the landing pad plasmid will remain between the recombination sites and the genomic sequence. In this case, the sequence derived from the second GOI plasmid will be flanked by sequences derived from the landing pad plasmid, which will be flanked by sequences derived from the parental plasmid.

[0150] From these explanations it follows that throughout this application references to inserting a plasmid into another plasmid generally do not necessarily require that one plasmid is completely replaced by another plasmid. Instead, one plasmid is completely or partially replaced by another plasmid, or an excised plasmid is completely or partially excised.

[0151] In some aspects, the present disclosure provides a method of generating an expressing cell, the method comprising: (a) integrating a second GOI plasmid (e.g., a linear plasmid) into the genome of a landing pad cell of the present disclosure at a targeted integration site using a site-specific recombinase, wherein the expression plasmid (P4) comprises (1) a polynucleotide sequence containing a nucleic acid encoding a GOI; and (2) two SSRS flanking the polynucleotide of (1); The site-specific recombinase recombination site of the landing pad plasmid is recombined with the corresponding site-specific recombinase recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid inserted into the genomic DNA of the landing pad cell.

[0152] The present disclosure also provides a method of generating an expressing cell, the method comprising: (a) integrating a second GOI plasmid (e.g., a linearized plasmid) into the genome of a parent cell comprising the parent plasmid at a targeted integration site using homologous recombination, wherein the expression plasmid comprises (1) a polynucleotide sequence containing a nucleic acid encoding a GOI; and (2) two SSRS flanking the polynucleotide of (1); The site-specific recombinase recombination site of the parent plasmid is recombined with the corresponding site-specific recombinase recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the parent plasmid inserted into the genomic DNA of the parent cell.

[0153] The present disclosure also provides a method of generating an expressing cell, the method comprising: integrating a second GOI plasmid (e.g., a linearized plasmid) into the genome of a parent cell comprising the parent plasmid at a targeted integration site using homologous recombination, wherein the resulting expression plasmid comprises a polynucleotide sequence comprising a nucleic acid encoding the GOI; and The parent plasmid and the GOI plasmid are recombined, whereby the GOI plasmid is integrated at an internal location within the parent plasmid that is inserted into the genomic DNA of the parent cell.

[0154] The present disclosure also provides a method of generating an expressing cell, the method comprising: integrating a second GOI plasmid (e.g., a linearized plasmid) into the genome of the parent cell containing the parent plasmid at a targeted integration site using homologous recombination, The parental plasmid recombines with the flanking genomic sequences, thereby integrating the GOI plasmid within the parental plasmid that was inserted into the genomic DNA of the parental cell.

[0155] It will be appreciated that while the methods disclosed herein involve two nested nuclease-mediated recombination events (e.g., homologous recombination between a parental plasmid and a landing pad plasmid in a parental cell, and site-specific recombinase recombination between a landing pad plasmid and a second GOI plasmid in a landing pad cell), other combinations of recombination events will be equally applicable, e.g., a first homologous recombination event between P1 and P2, and a second homologous recombination between P2 and P3.

[0156] It should also be understood that the teachings related to the integration of a plasmid (e.g., a landing pad plasmid or a GOI plasmid) in the context of the present disclosure are intended to cover the insertion of multiple plasmids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), which may be the same or different and which may differ with respect to their orientation in the final construct (e.g., whether each plasmid in the final construct is in a 5'-3' orientation or a 3'-5' orientation with respect to the original construct and the other plasmids in the final construct).

[0157] In some aspects, the present disclosure also provides a method of generating an expressing cell, the method comprising: integrating a GOI plasmid (e.g., a linear plasmid) into the genome of a parent cell using homologous recombination, wherein the expression plasmid comprises a polynucleotide sequence comprising a nucleic acid encoding the GOI; and Wherein the GOI plasmid is integrated at the position determined to correspond to the hot spot via homologous recombination. In some aspects, at least a portion of the parent plasmid is removed. In some aspects, the entire parent plasmid is removed.

[0158] In some aspects, the present disclosure also provides methods for identifying starting cells (parental cell lines) in an efficient manner to prepare landing pad cell lines capable of producing high titers. Therefore, the present disclosure provides methods for selecting parental cells to generate expression cells, for example, as disclosed in Examples 1 and 2.

[0159] In some aspects, the methods disclosed herein include removing at least a portion of at least one parent plasmid and introducing one or more landing pad plasmids having a landing pad, or a portion thereof, into the genome of the cell. In some aspects, the methods disclosed herein include removing only a portion of at least one parent plasmid and introducing one or more landing pad plasmids having a landing pad, or a portion thereof, into the genome of the cell.

[0160] In some aspects, methods for selecting a parental cell line suitable for developing a landing cell line of the present disclosure include: (i) selecting a cell line with a high expression titer of the target gene; (ii) further selecting cells having a low copy number of the ORF encoding the gene of interest. In some aspects, the parental cell has one or two copies of the ORF encoding the gene of interest. In some aspects, the parental cell has more than two copies of the ORF encoding the gene of interest.

[0161] In some aspects, the method for selecting a landing pad cell line includes screening for the loss of a parent plasmid or a portion thereof and selecting cells with such a loss (deletion). In some aspects, the method for selecting a parent cell line further includes screening for the presence of a landing pad, and selecting cells in which a landing pad is present. In some aspects, the method further includes screening a landing pad for the following features: the presence or absence of a low complexity region or a high complexity region, the presence or absence of a retrotransposon sequence, the presence or absence of an Alu repeat sequence, the presence or absence of a long interspersed nuclear element (LINE), the presence or absence of an island, the level of cytosine methylation, the level of histone acetylation, the presence or absence of an ORF and any combination thereof.

[0162] In some aspects, the cell is a CHO cell. In some aspects, the hotspot position comprises a sequence selected from the group consisting of SEQ ID NO: 18 or a fragment thereof and SEQ ID NO: 19 or a fragment thereof. In some aspects, the hotspot position comprises a sequence selected from the group consisting of SEQ ID NO: 114 or a fragment thereof and SEQ ID NO: 115 or a fragment thereof.

[0163] In some aspects, the GOI plasmid is inserted via homologous recombination or random integration at a position within the genomic sequence of SEQ ID NO: 18. In some aspects, the GOI plasmid is inserted via homologous recombination or random integration at a position within the genomic sequence of SEQ ID NO: 19. In some aspects, the GOI plasmid is inserted via homologous recombination or random integration at a position within the genomic sequence of SEQ ID NO: 114. In some aspects, the GOI plasmid is inserted via homologous recombination or random integration at a position within the genomic sequence of SEQ ID NO: 115.

[0164] In some aspects, the GOI plasmid is integrated via homologous recombination at a position within the genomic sequence wherein the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 18 or a subsequence thereof and / or the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 19 or a subsequence thereof.

[0165] In some aspects, the GOI plasmid is integrated via homologous recombination at a position within the genomic sequence wherein the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 114 or a subsequence thereof and / or the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 115 or a subsequence thereof.

[0166] In some aspects, the 5' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440 At least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 570, at least about 580, at least about 590, or at least about 600 consecutive nucleotides.

[0167] In some aspects, the 3' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440 At least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 570, at least about 580, at least about 590, or at least about 600 consecutive nucleotides.

[0168] In some aspects, the 5' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430 at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 570, at least about 580, at least about 590, or at least about 600 consecutive nucleotides, and the 3' homologous recombination site comprises a nucleotide sequence from SEQ ID NO: 19 or SEQ ID NO: NO: 115 is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, At least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 570, at least about 580, at least about 590, or at least about 600 consecutive nucleotides.

[0169] In some aspects, the GOI is an antibody. In some aspects, the GOI comprises the heavy chain (HC) of an antibody. In some aspects, the GOI comprises the light chain (LC) of an antibody. In some aspects, the GOI comprises the HC and LC of an antibody. In some aspects, the GOI comprises the antigen binding portion of an antibody. In some aspects, the expression plasmid comprises one, two or more copies of the GOI. In some aspects, the expression plasmid comprises one, two or more expression cassettes. In some aspects, the expression plasmid is bicistronic. In some aspects, the expression plasmid is polycodon.

[0170] In some aspects, the expression plasmid is integrated into the genome of the expression cell with at least one (1) copy number. In some aspects, the expression plasmid is integrated into the genome of the expression cell with one (1) copy number. In other aspects, the expression plasmid is integrated into the genome of the expression cell with a copy number of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 copies. In some aspects, there are more than 30 copies in the genome of the expression cell.

[0171] In other aspects, the expression plasmid is integrated into the genome of the expressing cell at a copy number of about 1 to about 3, about 3 to about 6, about 6 to about 9, about 9 to about 12, about 12 to about 15, about 15 to about 18, about 18 to about 21, about 21 to about 24, about 24 to about 27, about 27 to about 30, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 1 to about 10, about 5 to about 15, about 10 to about 20, about 15 to about 25, about 20 to about 30, about 1 to about 15, about 5 to about 20, about 10 to about 25, about 15 to about 30, about 1 to about 20, about 5 to about 25, about 10 to about 30 copies.

[0172] In some aspects, the methods disclosed herein comprise introducing a second GOI plasmid (P3; see e.g. Figure 5A ) was targeted for integration into the landing pad cell line to generate an expression plasmid (P4; see e.g. Figure 5A) and then measuring the expression of the GOI produced by the host cells. In some aspects, the expression level is measured quantitatively. In other aspects, the expression is measured qualitatively. The expression of the GOI can be measured by using any of the following methods known in the art: for example, cell sorting, FACS, cell surface staining, Western blot, Northern blot, column chromatography, capillary electrophoresis, microfluidics, UV absorbance, cell size, secreted protein levels, transcript levels, immunohistochemistry or any combination thereof.

[0173] In the context of the present disclosure, the recombinant expression level of the second GOI may correspond to expression from a single expression cassette, or to expression from multiple expression cassettes from an expression cell generated using the methods according to the present disclosure. In some aspects, expression of a GOI may correspond to multiple cassettes comprising the GOI inserted into the same site.

[0174] The present disclosure provides landing pad cells and expression cells produced according to the methods disclosed herein. In some aspects, the recombinant protein expression level of a second GOI (e.g., a second biological product, such as a second antibody) obtained when using the expression cells generated according to the methods of the present disclosure is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about %, at least about 190%, at least about 195%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%.

[0175] The present disclosure provides landing pad cells and expression cells produced according to the methods disclosed herein. In some aspects, the recombinant protein expression level of a second GOI (e.g., a second biological product, such as a second antibody) obtained when using the expression cells generated according to the methods of the present disclosure is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 15 ... About 110%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000% or more than 1000%.

[0176] The present disclosure provides landing pad cells and expression cells produced according to the methods disclosed herein. In some aspects, the recombinant protein expression level of a second GOI (e.g., a second biological product, such as a second antibody) obtained when using an expression cell generated according to the methods of the present disclosure is about 50% to about 55%, about 55% to about 60%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 125%, about 125% to about 130%, about 130% to about 135%, about 135% to about 140%, about 145% to about 146%, about 147% to about 148%, about 149% to about 150%, about 151% to about 152%, about 153% to about 154%, about 155% to about 156%, about 157% to about 158%, about 159% to about 160%, about 161% to about 162%, about 163% to about 164%, about 165% to about 166%, about 167% to about 168%, about 169% to about 170%, about 171% to about 172%, about 173% to about 174%, about 175% to about 176%, about 177% to about 178%, about 179% to about 180%, about 181% to about 181%, about 182% to about 183%, about 184% to about 185%, about % to about 140%, about 140% to about 145%, about 145% to about 150%, about 150% to about 155%, about 155% to about 160%, about 160% to about 165%, about 165% to about 170%, about 170% to about 175%, about 175% to about 180%, about 180% to about 185%, about 185% to about 190%, about 190% to about 195%, about 195% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, about 500% to about 600%, about 600% to about 700%, about 700% to about 800%, about 800% to about 900%, about 900% to about 1000%, or more than 1000%.

[0177] In some aspects of the present disclosure, the cells disclosed herein can be established as a cell line (i.e., a cell culture developed from a single cell) and therefore consist of cells with a uniform genetic composition, wherein under certain conditions the cells proliferate indefinitely in the laboratory, and in the case of expression cell lines, one or more genes of interest are stably integrated into the genome of the cells.

[0178] In some aspects, the targeted integration site is located within a "Chr3 TI contig," or chromosome 3 targeted integration locus, defined as a polynucleotide from chromosome 3 of Cricetulus griseus (Chinese hamster) comprising (i) a sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, or at least about 96.6% identical to SEQ ID NO:23 (5 kb sequence at the 5' end of a 26 Mb contig from gi|1497155598|ref|NW_020822499.1) at the 5' end of the polynucleotide, and (ii) a sequence at the 3' end of the polynucleotide that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, or at least about 96.6% identical to SEQ ID NO:23 (5 kb sequence at the 5' end of a 26 Mb contig from gi|1497155598|ref|NW_020822499.1) at the 5' end of the polynucleotide. NO:24 (3' 5 kb sequence from gi|1497155598|ref|NW_020822499.126 Mb contig) is at least 96.6% identical to the sequence, wherein the length of the polynucleotide is between 25 Mb (megabases) and 26.5 Mb (megabases).

[0179] In some aspects, the targeted integration site is located within a "Chr5 TI contig" or chromosome 5 targeted integration locus defined as a polynucleotide from chromosome 5 of C. cristae (Chinese hamster), comprising (i) a sequence at the 5' end of the polynucleotide that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, or at least about 96.6% identical to SEQ ID NO: 119 (5 kb sequence from the 5' end of the NW_020822577.1 18 Mb contig) and (ii) a sequence at the 3' end of the polynucleotide that is at least 96.6% identical to SEQ ID NO: 120 (5 kb sequence from the 3' end of the NW_020822577.1 18 Mb contig), wherein the length of the polynucleotide is between 17 Mb (megabases) and 19 Mb (megabases).

[0180] Actively transcribed genes described in the CHO RNA-seq dataset (see Singh et al. Biotechnol. 1.1001) were present within the hotspot of SEQ ID NO:22 (Refseq NW_020822499.1; available at ncbi.nlm.nih.gov / nuccore / NW_020822499.1?report=genbank and ncbi.nlm.nih.gov / nuccore / NW_020822499.1?report=fasta) and the hotspot of SEQ ID NO:118 (Refseq NW_020822577.1; available at ncbi.nlm.nih.gov / nuccore / NW_020822577.1?report=genbank and ncbi.nlm.nih.gov / nuccore / NW_020822577.1?report=fasta). J.2018Oct;13(10):e1800070, and Lin et al. PLoS Comput Biol 16(12):e1008498, which are incorporated herein by reference in their entirety) and confirmed by the applicant through experiments. For the first hotspot of NW_020822499.1, the closest gene on the 5' side of the deletion where the landing pad is located is Prkg1, 269 kb upstream, and the closest gene on the 3' side of the deletion where the landing pad is located is Mbl2, 43 kb downstream. No other active transcripts were identified between Prkg1 and Mbl2 by the applicant and in the CHO RNA-seq data set. For the second hotspot of NW_020822577.1, the closest gene on the 5' side of the deletion where the landing pad is located is Ackr1, 209 kb upstream, and the closest gene on the 3' side of the deletion where the landing pad is located is Crp, 170 kb downstream. No other active transcripts were identified between Ackr1 and Crp by the applicant and in the CHO RNA-seq data set. In some aspects, the targeted integration site is located at a position within SEQ ID NO:22 or SEQ ID NO:118 that does not affect the expression of one or more active transcribed genes. In some aspects, one or more active transcribed genes are located within the hotspot of SEQ ID NO:22 or the hotspot of SEQ ID NO:118. In some aspects, one or more active transcribed genes are located near the 5' end of the hotspot of SEQ ID NO:22 or the hotspot of SEQ ID NO:118. In some aspects, one or more active transcribed genes are located near the hotspot of SEQ ID NO:22 or the hotspot of SEQ ID NO:118.In some aspects of the disclosure, an actively transcribed gene is considered to be close to the 5' end or 3' end of a hotspot disclosed herein when it is located at a distance of about 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 75 kb, 100 kb, 125 kb, 150 kb, 175 kb, 200 kb, 225 kb, 250 kb, 275 kb, 300 kb, 325 kb, 350 kb, 375 kb, 400 kb, 425 kb, 450 kb, 475 kb or 500 kb from the 5' end or 3' end of a hotspot disclosed herein.

[0181] In some aspects, the targeted integration site is located at a specific position of the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig. In some aspects, the targeted integration site is located at a specific position in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig, within nucleotide positions 1 (5' start position) and 26,290,500 (3' end position) of SEQ ID NO: 22.

[0182] In some aspects, the targeted integration site is located at a specific position of the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is located at a specific position in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig, within nucleotide position 1 (5' start position) and 18,231,092 (3' end position) of SEQ ID NO: 118.

[0183] As used herein, the term "specific position" refers to a specific position (e.g., a single base) in the sequence shown, for example, in SEQ ID NO: 22 or the Chr3 TI contig, or in SEQ ID NO: 118 or the Chr5 TI contig, where integration will occur; for example, a specific position at position 100 would mean that integration will occur by insertion between nucleotides 100 and 101. In some aspects, the term "specific position" refers to a specific range of nucleotides between two positions that will be excised when integration occurs; for example, a specific position between positions 100 and 200 would mean that the original sequence comprising nucleotides 101 to 199 would be deleted and replaced by the integrated sequence.

[0184] In some aspects, the targeted integration site is located between the positions in the sequence shown in SEQ ID NO: 22 (corresponding to an exemplary targeted integration site of SEQ ID NO: 21) or Chr3 TI contigs or in SEQ ID NO: 118 (corresponding to an exemplary targeted integration site of SEQ ID NO: 117) or Chr5 TI contigs. In some aspects, the framed sequence (i.e., the sequence corresponding to the targeted integration site) is replaced by an expression plasmid (e.g., a parental plasmid or a landing pad plasmid as described herein). In some aspects, an expression plasmid (e.g., a parental plasmid or a landing pad plasmid as described herein) is integrated on the negative strand corresponding to the sequence shown in SEQ ID NO: 22 or Chr3 TI contigs or in SEQ ID NO: 118 or Chr5 TI contigs. Thus, in some aspects, the underlined sequences upstream (5') and downstream (3') of the boxed sequence correspond to the 3' and 5' junctions, respectively, of an integrated expression plasmid integrated on the negative strand corresponding to the sequence shown in SEQ ID NO:22 or the Chr3TI contig or in SEQ ID NO:118 or the Chr5 TI contig.

[0185] In some aspects, the targeted integration site is between positions 1 and 1,000,000; between positions 1,000,000 and 2,000,000; between positions 2,000,000 and 3,000,000; between positions 3,000,000 and 4,000,000; between positions 4,000,000 and 5,000,000; between positions 5,000,000 and 6,000,000; between positions 6,000,000 and 7,000,000; Between positions 7,000,000 and 8,000,000; Between positions 8,000,000 and 9,000,000; Between positions 9,000,000 and 10,000,000; Between positions 10,000,000 and 11,000,000; Between positions 11,000,000 and 12,000,000; Between positions 12,000,000 and 13,000,000; Between positions 13,000,000 and 14,000,000 00; between 14,000,000 and 15,000,000; between 15,000,000 and 16,000,000; between 16,000,000 and 17,000,000; between 17,000,000 and 18,000,000; between 18,000,000 and 19,000,000; between 19,000,000 and 20,000,000; between 20,000,000 between positions 0 and 21,000,000; between positions 21,000,000 and 22,000,000; between positions 22,000,000 and 23,000,000; between positions 23,000,000 and 24,000,000; between positions 24,000,000 and 25,000,000; between positions 25,000,000 and 26,000,000; or between positions 26,000,000 and 26,294,056.

[0186] In some aspects, the targeted integration site is in SEQ ID NO: 22 or Chr3 TI contig or in SEQ ID NO: 118 or Chr5 Between the following positions of the sequence shown in the TI contig: 1-100,000; 100,000-200,000; 200,000-300,000; 300,000-400,000; 400,000-500,000; 500,000-600,000; 600,000-700,000; 700,000-800,000; 800,000-900,000; 900,000-1,000,000; 1,000,000-1,100,000; 1,100,000-1,200,000; 1,200,000-1,300,000 0; 1,300,000-1,400,000; 1,400,000-1,500,000, 1,500,000-1,600,000; 1,600,000-1,700,000; 1,700,000-1,800,000; 1,800,000-1,900,000; 1,900,000-2,000,000; 2,000,000-2,100,000; 2,100,000-2,200,000; 2,200,000-2,300,000; 2,300,000-2,400,000; 2,400,000-2, 500,000, 2,500,000-2,600,000; 2,600,000-2,700,000; 2,700,000-2,800,000; 2,800,000-2,900,000; 2,900,000-3,000,000; 3,000,000-3,100,000; 3,100,000-3,200,000; 3,200,000-3,300,000; 3,300,000-3,400,000; 3,400,000-3,500,000, 3,500,000-3,600,000; 3,600, 3,700,000-3,800,000; 3,800,000-3,900,000; 3,900,000-4,000,000; 4,000,000-4,100,000; 4,100,000-4,200,000; 4,200,000-4,300,000; 4,300,000-4,400,000; 4,400,000-4,500,000, 4,500,000-4,600,000; 4,600,000-4,700,000; 4,700,000-4,800,000;4,800,000-4,900,000；4,900,000-5,000,000；5,000,000-5,100,000；5,100,000-5,200,000；5,200,000-5,300,000；5,300,000-5,400,000；5,400,000-5,500,000、5,500,000-5,600,000；5,600,000-5,700,000；5,700,000-5,800,000；5,800,000-5,900,000；5,900,000-6,000,000；6,000,000-6,100,000；6,100,000-6,200,000；6,200,000-6,300,000；6,300,000-6,400,000；6,400,000-6,500,000、6,500,000-6,600,000；6,600,000-6,700,000；6,700,000-6,800,000；6,800,000-6,900,000；6,900,000-7,000,000；7,000,000-7,100,000；7,100,000-7,200,000；7,200,000-7,300,000；7,300,000-7,400,000；7,400,000-7,500,000、7,500,000-7,600,000；7,600,000-7,700,000；7,700,000-7,800,000；7,800,000-7,900,000；7,900,000-8,000,000；8,000,000-8,100,000；8,100,000-8,200,000；8,200,000-8,300,000；8,300,000-8,400,000；8,400,000-8,500,000、8,500,000-8,600,000；8,600,000-8,700,000；8,700,000-8,800,000；8,800,000-8,900,000；8,900,000-9,000,000；9,000,000-9,100,000；9,100,000-9,200,000；9,200,000-9,300,000；9,300,000-9,400,000；9,400,000-9,500,000、9,500,000-9,600,000；9,600,000-9,700,000；9,700,000-9,800,000；9,800,000-9,900,000；9,900,000-10,000,000；10,000,000-10,100,000；10,100,000-10,200,000；10,200,000-10,300,000；10,300,000-10,400,000；10,400,000-10,500,000、10,500,000-10,600,000；10,600,000-10,700,000；10,700,000-10,800,000；10,800,000-10,900,000；10,900,000-11,000,000；11,000,000-11,100,000；11,100,000-11,200,000；11,200,000-11,300,000；11,300,000-11,400,000；11,400,000-11,500,000、11,500,000-11,600,000；11,600,000-11,700,000；11,700,000-11,800,000；11,800,000-11,900,000；11,900,000-12,000,000；12,000,000-12,100,000；12,100,000-12,200,000；12,200,000-12,300,000；12,300,000-12,400,000；12,400,000-12,500,000、12,500,000-12,600,000；12,600,000-12,700,000；12,700,000-12,800,000；12,800,000-12,900,000；12,900,000-13,000,000；13,000,000-13,100,000；13,100,000-13,200,000；13,200,000-13,300,000；13,300,000-13,400,000；13,400,000-13,500,000、13,500,000-13,600,000；13,600,000-13,700,000；13,700,000-13,800,000；13,800,000-13,900,000；13,900,000-14,000,000；14,000,000-14,100,000；14,100,000-14,200,000；14,200,000-14,300,000；14,300,000-14,400,000；14,400,000-14,500,000、14,500,000-14,600,000；14,600,000-14,700,000；14,700,000-14,800,000；14,800,000-14,900,000；14,900,000-15,000,000；15,000,000-15,100,000；15,100,000-15,200,000；15,200,000-15,300,000；15,300,000-15,400,000；15,400,000-15,500,000、15,500,000-15,600,000；15,600,000-15,700,000；15,700,000-15,800,000；15,800,000-15,900,000；15,900,000-16,000,000；16,000,000-16,100,000；16,100,000-16,200,000；16,200,000-16,300,000；16,300,000-16,400,000；16,400,000-16,500,000、16,500,000-16,600,000；16,600,000-16,700,000；16,700,000-16,800,000；16,800,000-16,900,000；16,900,000-17,000,000；17,000,000-17,100,000；17,100,000-17,200,000；17,200,000-17,300,000；17,300,000-17,400,000；17,400,000-17,500,000、17,500,000-17,600,000；17,600,000-17,700,000；17,700,000-17,800,000；17,800,000-17,900,000；17,900,000-18,000,000；18,000,000-18,100,000；18,100,000-18,200,000；18,200,000-18,300,000；18,300,000-18,400,000；18,400,000-18,500,000、18,500,000-18,600,000；18,600,000-18,700,000；18,700,000-18,800,000；18,800,000-18,900,000；18,900,000-19,000,000；19,000,000-19,100,000；19,100,000-19,200,000；19,200,000-19,300,000；19,300,000-19,400,000；19,400,000-19,500,000、19,500,000-19,600,000；19,600,000-19,700,000；19,700,000-19,800,000；19,800,000-19,900,000；19,900,000-20,000,000；20,000,000-20,100,000；20,100,000-20,200,000；20,200,000-20,300,000；20,300,000-20,400,000；20,400,000-20,500,000、20,500,000-20,600,000；20,600,000-20,700,000；20,700,000-20,800,000；20,800,000-20,900,000；20,900,000-21,000,000；21,000,000-21,100,000；21,100,000-21,200,000；21,200,000-21,300,000；21,300,000-21,400,000；21,400,000-21,500,000、21,500,000-21,600,000；21,600,000-21,700,000；21,700,000-21,800,000；21,800,000-21,900,000；21,900,000-22,000,000；22,000,000-22,100,000；22,100,000-22,200,000；22,200,000-22,300,000；22,300,000-22,400,000；22,400,000-22,500,000、22,500,000-22,600,000；22,600,000-22,700,000；22,700,000-22,800,000；22,800,000-22,900,000；22,900,000-23,000,000；23,000,000-23,100,000；23,100,000-23,200,000；23,200,000-23,300,000；23,300,000-23,400,000；23,400,000-23,500,000、23,500,000-23,600,000；23,600,000-23,700,000；23,700,000-23,800,000；23,800,000-23,900,000；23,900,000-24,000,000；24,000,000-24,100,000；24,100,000-24,200,000；24,200,000-24,300,000；24,300,000-24,400,000；24,400,000-24,500,000、24,500,000-24,600,000；24,600,000-24,700,000；24,700,000-24,800,000；24,800,000-24,900,000；24,900,000-25,000,000；25,000,000-25,100,000；25,100,000-25,200,000；25,200,000-25,300,000；25,300,000-25,400,000；25,400,000-25,500,000、25,500,000-25,600,000；25,600,000-25,700,000；25,700,000-25,800,000；25,800,000-25,900,000；25,900,000-26,000,000；26,000,000-26,100,000；26,100,000-26,200,000；26,200,000-26,294,056。；

[0187] In some aspects, the targeted integration site is set forth above in SEQ ID NO: 22 or Chr3 TI contig or in SEQ ID NO: 118 or Chr5 The range of sequences shown in the Ti contigs is between any of the following positions: 1-1,000; 1,000-2,000; 2,000-3,000; 3,000-4,000; 4,000-5,000; 5,000-6,000; 6,000-7,000; 7,000-8,000; 8,000-9,000; 9,000-10,000; 10,000-11,000; 11,000-12,000; 12,000-13,000; 13,000-14,000; 14 ,000-15,000; 15,000-16,000; 16,000-17,000; 17,000-18,000; 18,000-19,000; 19,000-20,000; 20,000-21,000; 21,000-22,000; 22,000-23,000; 23,000-24,000; 24,000-25,000; 25,000-26,000; 26,000-27,000; 27,000-28,000; 28,000-29,000; 29,000-30,000; 30,000-31,0 00; 31,000-32,000; 32,000-33,000; 33,000-34,000; 34,000-35,000; 35,000-36,000; 36,000-37,000; 37,000-38,000; 38,000-39,000; 39,000-40,000; 40,000-41,000; 41,000-42,000; 42,000-43,000; 43,000-44,000; 44,000-45,000; 45,000-46,000; 46,000-47,000; 47,00 0-48,000; 48,000-49,000; 49,000-50,000; 50,000-51,000; 51,000-52,000; 52,000-53,000; 53,000-54,000; 54,000-55,000; 55,000-56,000; 56,000-57,000; 57,000-58,000; 58,000-59,000; 59,000-60,000; 60,000-61,000; 61,000-62,000; 62,000-63,000; 63,000-64,000;64,000-65,000; 65,000-66,000; 66,000-67,000; 67,000-68,000; 68,000-69,000; 69,000-70,000; 70,000-71,000; 71,000-72,000; 72,000-73,000; 73,000-74,000; 74,000-75,000; 75,000-76,000; 76,000-77,000; 77,000-78,000; 78,000-79,000; 79,000-80,000; 80,000-81,000; 81,000-82,000; 8 2,000-83,000; 83,000-84,000; 84,000-85,000; 85,000-86,000; 86,000-87,000; 87,000-88,000; 88,000-89,000; 89,000-90,000; 90,000-91,000; 9 1,000-92,000; 92,000-93,000; 93,000-94,000; 94,000-95,000; 95,000-96,000; 96,000-97,000; 97,000-98,000; 98,000-99,000; or 99,000-100,000. ;

[0188] In some aspects, the targeted integration site is set forth above in SEQ ID NO: 22 or Chr3 TI contig or in SEQ ID NO: 118 or Chr5 Any of the following positions of a subrange of sequences 1-1,000 to 99,000-100,000 represented in the TI contigs: 1-10; 10-20; 20-30; 30-40; 40-50; 50-60; 60-70; 70-80; 80-90; 90-100; 100-110; 110-120; 120-130; 130-140; 140-150; 150-160; 160-170; 170-180; 180-190; 190-200; 200-210; 210-220; 220-230; 230-240; 240-250 0; 250-260; 260-270; 270-280; 280-290; 290-300; 300-310; 310-320; 320-330; 330-340; 340-350; 350-360; 360-370; 370-380; 380-390; 390-400; 400-410; 410-420; 420-430; 430-440; 440-450; 450-460; 460-470; 470-480; 480-490; 490-500; 500-510; 510-520; 520-530; 53 0-540; 540-550; 550-560; 560-570; 570-580; 580-590; 590-600; 600-610; 610-620; 620-630; 630-640; 640-650; 650-660; 660-670; 670-680; 680-690; 690-700; 700-710; 710-720; 720-730; 730-740; 740-750; 750-760; 760-770; 770-780; 780-790; 790-800; 800-810; 810-8 20; 820-830; 830-840; 840-850; 850-860; 860-870; 870-880; 880-890; 890-900; 900-910; 910-920; 920-930; 930-940; 940-950; 950-960; 960-970; 970-980; 980-990; 990-1000; 1-100; 100-200; 200-300; 300-400; 400-500; 500-600; 600-700; 700-800; 800-900; or 900-1000.

[0189] In some aspects, the targeted integration site is located between the following positions upstream or downstream of the sequence shown in SEQ ID NO:21 or SEQ ID NO:117: about 1 to about 10; about 10 to about 20; about 20 to about 30; about 30 to about 40; about 40 to about 50; about 50 to about 60; about 60 to about 70; about 70 to about 80; about 80 to about 90; about 90 to about 100; about 100 to about 110; about 110 to about 120; about 120 to about 130; about 130 to about 140; about 140 to about 150; about 150 to about 160; about 160 to about 170; about 170 to about 180; about 180 to about 190; about 190 to about 200; about 200 to about 210; about 210 to about 220; about 220 to about 230; about 230 to about about 240 to about 250; about 250 to about 260; about 260 to about 270; about 270 to about 280; about 280 to about 290; about 290 to about 300; about 300 to about 310; about 310 to about 320; about 320 to about 330; about 330 to about 340; about 340 to about 350; about 350 to about 360; about 360 to about 370; about 370 to about 380; about 380 to about 390; about 390 to about 400; about 400 to about 410; about 410 to about 420; about 420 to about 430; about 430 to about 440; about 440 to about 450; about 450 to about 460; about 460 to about 470; about 470 to about 480 about 500 to about 510; about 510 to about 520; about 520 to about 530; about 530 to about 540; about 540 to about 550; about 550 to about 560; about 560 to about 570; about 570 to about 580; about 580 to about 590; about 590 to about 600; about 600 to about 610; about 610 to about 620; about 620 to about 630; about 630 to about 640; about 640 to about 650; about 650 to about 660; about 660 to about 670; about 670 to about 680; about 680 to about 690; about 690 to about 700; about 700 to about 710; about 710 to about 720 0; about 720 to about 730; about 730 to about 740; about 740 to about 750; about 750 to about 760; about 760 to about 770; about 770 to about 780; about 780 to about 790; about 790 to about 800; about 800 to about 810; about 810 to about 820; about 820 to about 830; about 830 to about 840; about 840 to about 850; about 850 to about 860; about 860 to about 870; about 870 to about 880; about 880 to about 890; about 890 to about 900; about 900 to about 910; about 910 to about 920; about 920 to about 930; about 930 to about 940; about 940 to about 950; about 950 to about 960;about 960 to about 970; about 970 to about 980; about 980 to about 990; about 990 to about 1000; about 1,000 to about 2,000; about 2,000 to about 3,000; about 3,000 to about 4,000; about 4,000 to about 5,000; about 5,000 to about 6,000; about 6,000 to about 7,000; about 7,000 to about 8,000; about 8,000 to about 9,000; about 9,000 to about 10,000; about 10,000 to about 11,000; about 11,000 to about 12,000; about 12,000 to about 13,000; about 13,000 to about 14,000; about 14,000 to about 15,000; about 1 5,000 to about 16,000; about 16,000 to about 17,000; about 17,000 to about 18,000; about 18,000 to about 19,000; about 19,000 to about 20,000; about 20,000 to about 21,000; about 21,000 to about 22,000; about 22,000 to about 23,000; about 23,000 to about 24,000; about 24,000 to about 25,000; about 25,000 to about 26,000; about 26,000 to about 27,000; about 27,000 to about 28,000; about 28,000 to about 29,000; about 29,000 to about 30,000; about 30,000 to about 31, about 31,000 to about 32,000; about 32,000 to about 33,000; about 33,000 to about 34,000; about 34,000 to about 35,000; about 35,000 to about 36,000; about 36,000 to about 37,000; about 37,000 to about 38,000; about 38,000 to about 39,000; about 39,000 to about 40,000; about 40,000 to about 41,000; about 41,000 to about 42,000; about 42,000 to about 43,000; about 43,000 to about 44,000; about 44,000 to about 45,000; about 45,000 to about 46,000; about 46,000 0 to about 47,000; about 47,000 to about 48,000; about 48,000 to about 49,000; about 49,000 to about 50,000; about 50,000 to about 51,000; about 51,000 to about 52,000; about 52,000 to about 53,000; about 53,000 to about 54,000; about 54,000 to about 55,000; about 55,000 to about 56,000; about 56,000 to about 57,000; about 57,000 to about 58,000; about 58,000 to about 59,000; about 59,000 to about 60,000; about 60,000 to about 61,000; about 61,000 to about 62,000;about 62,000 to about 63,000; about 63,000 to about 64,000; about 64,000 to about 65,000; about 65,000 to about 66,000; about 66,000 to about 67,000; about 67,000 to about 68,000; about 68,000 to about 69,000; about 69,000 to about 70,000; about 70,000 to about 71,000; about 71,000 to about 72,000; about 72,000 to about 73,000; about 73,000 to about 74,000; about 74,000 to about 75,000; about 75,000 to about 76,000; about 76,000 to about 77,000; about 77,000 to about 78,000; about 78,000 to about 79,000; about 79,000 to about 80,000; about 80,000 to about 81,000; about 81,000 to about 82,000; about 82,000 to about 83,000; about 83,000 to about 84,000; about 84,000 to about 85,000; about 85,000 to about 86,000; about 86,000 to about 87,000; about 87,000 to about 88,000; about 88,000 to about 89,000; about 89,000 to about 90,000; about 90,000 to about 91,000; about 91,000 to about 92,000; about 92,000 to about 93,000; about 93,000 to about 94,000; about 94,000 to about 95,000; about 95,000 to about 96,000; about 96,000 to about 97,000; about 97,000 to about 98,000; about 98,000 to about 99,000; about 99,000 to about 100,000; about 100,000 to about 200,000; about 200,000 to about 300,000; about 300,000 to about 400,000; about 400,000 to about 500,000; about 500,000 to about 600,000; about 600,000 to about 700,000; about 700,000 to about 800,000; about 800,000 to about about 900,000; about 900,000 to about 1,000,000; about 1,000,000 to about 1,100,000; about 1,100,000 to about 1,200,000; about 1,200,000 to about 1,300,000; about 1,300,000 to about 1,400,000; about 1,400,000 to about 1,500,000; about 1,500,000 to about 1,600,000; about 1,600,000 to about 1,700,000; about 1,700,000 to about 1,800,000; about 1,800,000 to about 1,900,000; about 1,900,000 to about 2,000,000;about 2,000,000 to about 2,100,000; about 2,100,000 to about 2,200,000; about 2,200,000 to about 2,300,000; about 2,300,000 to about 2,400,000; about 2,400,000 to about 2,500,000; about 2,500,000 to about 2,600,000; about 2,600,000 to about 2,700,000; about 2,700,000 to about 2,800,000; about 2,800,000 to about 2,900,000; about 2,900,000 to about 3,000,000; about 3,000,000 to about 3,100,000; about 3,100,000 0,000 to about 3,200,000; about 3,200,000 to about 3,300,000; about 3,300,000 to about 3,400,000; about 3,400,000 to about 3,500,000; about 3,500,000 to about 3,600,000; about 3,600,000 to about 3,700,000; about 3,700,000 to about 3,800,000; about 3,800,000 to about 3,900,000; about 3,900,000 to about 4,000,000; about 4,000,000 to about 4,100,000; about 4,100,000 to about 4,200,000; about 4,200,000 to about 4,300,000; about 4,300,000 to about 4,400,000; about 4,400,000 to about 4,500,000; about 4,500,000 to about 4,600,000; about 4,600,000 to about 4,700,000; about 4,700,000 to about 4,800,000; about 4,800,000 to about 4,900,000; about 4,900,000 to about 5,000,000; about 5,000,000 to about 5,100,000; about 5,100,000 to about 5,200,000; about 5,200,000 to about 5,300,000; about 5,300,000 to about 5,40 0,000; about 5,400,000 to about 5,500,000; about 5,500,000 to about 5,600,000; about 5,600,000 to about 5,700,000; about 5,700,000 to about 5,800,000; about 5,800,000 to about 5,900,000; about 5,900,000 to about 6,000,000; about 6,000,000 to about 6,100,000; about 6,100,000 to about 6,200,000; about 6,200,000 to about 6,300,000; about 6,300,000 to about 6,400,000; about 6,400,000 to about 6,500,000;about 6,500,000 to about 6,600,000; about 6,600,000 to about 6,700,000; about 6,700,000 to about 6,800,000; about 6,800,000 to about 6,900,000; about 6,900,000 to about 7,000,000; about 7,000,000 to about 7,100,000; about 7,100,000 to about 7,200,000; about 7,200,000 to about 7,300,000; about 7,300,000 to about 7,400,000; about 7,400,000 to about 7,500,000; about 7,500,000 to about 7,600,000; about 7,6 00,000 to about 7,700,000; about 7,700,000 to about 7,800,000; about 7,800,000 to about 7,900,000; about 7,900,000 to about 8,000,000; about 8,000,000 to about 8,100,000; about 8,100,000 to about 8,200,000; about 8,200,000 to about 8,300,000; about 8,300,000 to about 8,400,000; about 8,400,000 to about 8,500,000; about 8,500,000 to about 8,600,000; about 8,600,000 to about 8,700,000; about 8,700,000 0 to about 8,800,000; about 8,800,000 to about 8,900,000; about 8,900,000 to about 9,000,000; about 9,000,000 to about 9,100,000; about 9,100,000 to about 9,200,000; about 9,200,000 to about 9,300,000; about 9,300,000 to about 9,400,000; about 9,400,000 to about 9,500,000; about 9,500,000 to about 9,600,000; about 9,600,000 to about 9,700,000; about 9,700,000 to about 9,800,000; about 9,800,000 to about 9, about 10,000,000 to about 10,000,000; about 10,000,000 to about 10,100,000; about 10,100,000 to about 10,200,000; about 10,200,000 to about 10,300,000; about 10,300,000 to about 10,400,000; about 10,400,000 to about 10,500,000; about 10,500,000 to about 10,600,000; about 10,600,000 to about 10,700,000; about 10,700,000 to about 10,800,000; about 10,800,000 to about 10,900,000;about 10,900,000 to about 11,000,000; about 11,000,000 to about 11,100,000; about 11,100,000 to about 11,200,000; about 11,200,000 to about 11,300,000; about 11,300,000 to about 11,400,000; about 11,400,000 to about 11,500,000; about 11,500,000 to about 11,600,000; about 11,600,000 to about 11,700,000; about 11,700,000 to about 11,800,000; about 11,800,000 to about 11,900,000; about 11,900,000 0,000 to about 12,000,000; about 12,000,000 to about 12,100,000; about 12,100,000 to about 12,200,000; about 12,200,000 to about 12,300,000; about 12,300,000 to about 12,400,000; about 12,400,000 to about 12,500,000; about 12,500,000 to about 12,600,000; about 12,600,000 to about 12,700,000; about 12,700,000 to about 12,800,000; about 12,800,000 to about 12,900,000; about 12,900,000 to about 13,000,000; about 13,000,000 to about 13,100,000; about 13,100,000 to about 13,200,000; about 13,200,000 to about 13,300,000; about 13,300,000 to about 13,400,000; about 13,400,000 to about 13,500,000; about 13,500,000 to about 13,600,000; about 13,600,000 to about 13,700,000; about 13,700,000 to about 13,800,000; about 13,800,000 to about 13,900,000; about 13,900,000 to about 14,000 0,000; about 14,000,000 to about 14,100,000; about 14,100,000 to about 14,200,000; about 14,200,000 to about 14,300,000; about 14,300,000 to about 14,400,000; about 14,400,000 to about 14,500,000; about 14,500,000 to about 14,600,000; about 14,600,000 to about 14,700,000; about 14,700,000 to about 14,800,000; about 14,800,000 to about 14,900,000; about 14,900,000 to about 15,000,000;about 15,000,000 to about 15,100,000; about 15,100,000 to about 15,200,000; about 15,200,000 to about 15,300,000; about 15,300,000 to about 15,400,000; about 15,400,000 to about 15,500,000; about 15,500,000 to about 15,600,000; about 15,600,000 to about 15,700,000; about 15,700,000 to about 15,800,000; about 15,800,000 to about 15,900,000; about 15,900,000 to about 16,000,000; about 16,000,000 0,000 to about 16,100,000; about 16,100,000 to about 16,200,000; about 16,200,000 to about 16,300,000; about 16,300,000 to about 16,400,000; about 16,400,000 to about 16,500,000; about 16,500,000 to about 16,600,000; about 16,600,000 to about 16,700,000; about 16,700,000 to about 16,800,000; about 16,800,000 to about 16,900,000; about 16,900,000 to about 17,000,000; about 17,000,000 to about 17,100,000; about 17,100,000 to about 17,200,000; about 17,200,000 to about 17,300,000; about 17,300,000 to about 17,400,000; about 17,400,000 to about 17,500,000; about 17,500,000 to about 17,600,000; about 17,600,000 to about 17,700,000; about 17,700,000 to about 17,800,000; about 17,800,000 to about 17,900,000; about 17,900,000 to about 18,000,000; about 18,000,000 to about 18,10 0,000; about 18,100,000 to about 18,200,000; about 18,200,000 to about 18,300,000; about 18,300,000 to about 18,400,000; about 18,400,000 to about 18,500,000; about 18,500,000 to about 18,600,000; about 18,600,000 to about 18,700,000; about 18,700,000 to about 18,800,000; about 18,800,000 to about 18,900,000; about 18,900,000 to about 19,000,000; about 19,000,000 to about 19,100,000;about 19,100,000 to about 19,200,000; about 19,200,000 to about 19,300,000; about 19,300,000 to about 19,400,000; about 19,400,000 to about 19,500,000; about 19,500,000 to about 19,600,000; about 19,600,000 to about 19,700,000; about 19,700,000 to about 19,800,000; about 19,800,000 to about 19,900,000; about 19,900,000 to about 20,000,000; about 20,000,000 to about 20,100,000; about 20,10 0,000 to about 20,200,000; about 20,200,000 to about 20,300,000; about 20,300,000 to about 20,400,000; about 20,400,000 to about 20,500,000; about 20,500,000 to about 20,600,000; about 20,600,000 to about 20,700,000; about 20,700,000 to about 20,800,000; about 20,800,000 to about 20,900,000; about 20,900,000 to about 21,000,000; about 21,000,000 to about 21,100,000; about 21,100,000 to about 21,200,000; about 21,200,000 to about 21,300,000; about 21,300,000 to about 21,400,000; about 21,400,000 to about 21,500,000; about 21,500,000 to about 21,600,000; about 21,600,000 to about 21,700,000; about 21,700,000 to about 21,800,000; about 21,800,000 to about 21,900,000; about 21,900,000 to about 22,000,000; about 22,000,000 to about 22,100,000; about 22,100,000 to about 22,20 0,000; about 22,200,000 to about 22,300,000; about 22,300,000 to about 22,400,000; about 22,400,000 to about 22,500,000; about 22,500,000 to about 22,600,000; about 22,600,000 to about 22,700,000; about 22,700,000 to about 22,800,000; about 22,800,000 to about 22,900,000; about 22,900,000 to about 23,000,000; about 23,000,000 to about 23,100,000; about 23,100,000 to about 23,200,000;about 23,200,000 to about 23,300,000; about 23,300,000 to about 23,400,000; about 23,400,000 to about 23,500,000; about 23,500,000 to about 23,600,000; about 23,600,000 to about 23,700,000; about 23,700,000 to about 23,800,000; about 23,800,000 to about 23,900,000; about 23,900,000 to about 24,000,000; about 24,000,000 to about 24,100,000; about 24,100,000 to about 24,200,000; about 24,200,000 to about 24,300,000; about 24,300,000 to about 24,400,000; about 24,400,000 to about 24,500,000; about 24,500,000 to about 24,600,000; about 24,600,000 to about 24,700,000; about 24,700,000 to about 24,800,000; about 24,800,000 to about 24,900,000; about 24,900,000 to about 25,000,000; about 25,000,000 to about 25,100,000; about 25,100,000 to about 25,200,000; about 25,200,000 to about 25,300,000; About 25,300,000 to about 25,400,000; about 25,400,000 to about 25,500,000; about 25,500,000 to about 25,600,000; about 25,600,000 to about 25,700,000; about 25,700,000 to about 25,800,000; about 25,800,000 to about 25,900,000; about 25,900,000 to about 26,000,000. ;

[0190] In some aspects, the targeting is to about 10; at least about 20; at least about 30; at least about 40; at least about 50; at least about 60; at least about 70; at least about 80; at least about 90; at least about 100; at least about 110; at least about 120; at least about 130; at least about 140; at least about 150; at least about 160; at least about 170; at least about 180; at least about 190; at least about 200; at least about 210; at least about 220; at least about 230; at least about 240; at least about 250; at least about 260; at least about 270; at least about 280; at least about 290; at least about 300; at least about 310; at least about 320; at least about 330; at least about 340; at least about 350; at least about 360; at least about 370; at least about 380; at least about 390; at least about 400; at least about 410; at least about 420; at least about 430; at least about 440; at least about 450; at least about 460; at least about 470; at least about 480; at least about 490; at least about 500; at least about 510; at least about 520; at least about 530; at least about 540; at least about 550; at least about 560; at least about 570; at least about 580; at least about 590; at least about 600; at least about 610; at least about 620; at least about 630; at least about 640; at least about 650; at least about 660; at least about 670; at least about 680 80; at least about 690; at least about 700; at least about 710; at least about 720; at least about 730; at least about 740; at least about 750; at least about 760; at least about 770; at least about 780; at least about 790; at least about 800; at least about 810; at least about 820; at least about 830; at least about 840; at least about 850; at least about 860; at least about 870; at least about 880; at least about 890; at least about 900; at least about 910; at least about 920; at least about 930; at least about 940; at least about 950; at least about 960; at least about 970; at least about 980; at least about 990; at least about 1,000; at least about 2,000; at least about 3,000; at least about 4,000; at least about 5,000; at least about 6,000; at least about 7,000; at least about 8,000; at least about 9,000; at least about 10,000; at least about 11,000; at least about 12,000; at least about 13,000; at least about 14,000; at least about 15,000; at least about 16,000; at least about 17,000; at least about 18,000; at least about 19,000; at least about 20,000; at least about 21,000; at least about 22,000; at least about 23,000; at least about 24,000; at least about 25,000; at least about 26,000; at least about 27,000;at least about 28,000; at least about 29,000; at least about 30,000; at least about 31,000; at least about 32,000; at least about 33,000; at least about 34,000; at least about 35,000; at least about 36,000; at least about 37,000; at least about 38,000; at least about 39,000; at least about 40,000; at least about 41,000; at least about 42,000; at least about 43,000; at least about 44,000; at least about 45,000; at least about 46,000; at least about 47,000; at least about 48,000; at least about 49,000; at least about 50,000; at least about 51,000; at least about 52,000; at least about 53,000; at least about 54,000; at least about 55,000; at least about 56,000; at least about 57,000; at least about 58,000; at least about 59,000; at least about 60,000; at least about 61,000; at least about 62,000; at least about 63,000; at least about 64,000; at least about 65,000; at least about 66,000; at least about 67,000; at least about 68,000; at least about 69,000; at least about 70,000; at least about 71,000; at least about 72,000; at least about 73,000; at least about 74,000; at least about 75,000; at least about 76,000; at least about 77,000; at least about 78,000; at least about 79,000; at least about 80,000; at least about 81,000; at least about 82,000; at least about 83,000; at least about 84,000; at least about 85,000; at least about 86,000; at least about 87,000; at least about 88,000; at least about 89,000; at least about 90,000; at least about 91,000; at least about 92,000; at least about 93,000; at least about 94,000; at least about 95,000; at least about 96,000; at least about 97,000; at least about 98,000; at least about 99,000; at least about 100,000; at least about 200,000; at least about 300,000 00; at least about 400,000; at least about 500,000; at least about 600,000; at least about 700,000; at least about 800,000; at least about 900,000; at least about 1,000,000; at least about 1,100,000; at least about 1,200,000; at least about 1,300,000; at least about 1,400,000; at least about 1,500,000; at least about 1,600,000; at least about 1,700,000; at least about 1,800,000; at least about 1,900,000; at least about 2,000,000; at least about 2,100,000; at least about 2,200,000; at least about 2,300,000;at least about 2,400,000; at least about 2,500,000; at least about 2,600,000; at least about 2,700,000; at least about 2,800,000; at least about 2,900,000; at least about 3,000,000; at least about 3,100,000; at least about 3,200,000; at least about 3,300,000; at least about 3,400,000; at least about 3,500,000; at least about 3,600,000; at least about 3,700,000; at least about 3,800,000; at least about 3,900,000; at least about 4,000,000; at least about 4,100,000; at least about 4,200,000; at least about 4,300,000; at least about 4,400,000; at least about 4,500,000; at least about 4,600,000; at least about 4,700,000; at least about 4,800,000; at least about 4,900,000; at least about 5,000,000; at least about 5,100,000; at least about 5,200,000; at least about 5,300,000; at least about 5,400,000; at least about 5,500,000; at least about 5,600,000; at least about 5,700,000; at least about 5,800,000; at least about 5,900,000; at least about 6,000,000; at least about 6,100,000; at least about 6,200,000; at least about 6,300,000; at least about 6,400,000; at least about 6,500,000; at least about 6,600,000; at least about 6,700,000; at least about 6,800,000; at least about 6,900,000; at least about 7,000,000; at least about 7,100,000; at least about 7,200,000; at least about 7,300,000; at least about 7,400,000; at least about 7,500,000; at least about 7,600,000; at least about 7,700,000; at least about 7,800,000; at least about 7,900,000; at least about 8,000,000; at least about 8,100,000; at least about 8,200,000; at least about 8,300,000; at least about 8,400,000; at least about 8,500,000; at least about 8,600,000; at least about 8,700,000; at least about 8,800,000; at least about 8,900,000; at least about 9,000,000; at least about 9,100,000; at least about 9,200,000; at least about 9,300,000; at least about 9,400,000; at least about 9,500,000; at least about 9,600,000; at least about 9,700,000; at least about 9,800,000; at least about 9,900,000;at least about 10,000,000; at least about 10,100,000; at least about 10,200,000; at least about 10,300,000; at least about 10,400,000; at least about 10,500,000; at least about 10,600,000; at least about 10,700,000; at least about 10,800,000; at least about 10,900,000; at least about 11,000,000; at least about 11,100,000; at least about 11,200,000; at least about 11,300,000; at least about 11,400,000; at least about 11,500,000; at least about 11,600,000; at least about 11,700, 000; at least about 11,800,000; at least about 11,900,000; at least about 12,000,000; at least about 12,100,000; at least about 12,200,000; at least about 12,300,000; at least about 12,400,000; at least about 12,500,000; at least about 12,600,000; at least about 12,700,000; at least about 12,800,000; at least about 12,900,000; at least about 13,000,000; at least about 13,100,000; at least about 13,200,000; at least about 13,300,000; at least about 13,400,000; at least about 13,5 00,000; at least about 13,600,000; at least about 13,700,000; at least about 13,800,000; at least about 13,900,000; at least about 14,000,000; at least about 14,100,000; at least about 14,200,000; at least about 14,300,000; at least about 14,400,000; at least about 14,500,000; at least about 14,600,000; at least about 14,700,000; at least about 14,800,000; at least about 14,900,000; at least about 15,000,000; at least about 15,100,000; at least about 15,200,000; at least about 15,300,000; at least about 15,400,000; at least about 15,500,000; at least about 15,600,000; at least about 15,700,000; at least about 15,800,000; at least about 15,900,000; at least about 16,000,000; at least about 16,100,000; at least about 16,200,000; at least about 16,300,000; at least about 16,400,000; at least about 16,500,000; at least about 16,600,000; at least about 16,700,000; at least about 16,800,000; at least about 16,900,000; at least about 17,000,000;at least about 17,100,000; at least about 17,200,000; at least about 17,300,000; at least about 17,400,000; at least about 17,500,000; at least about 17,600,000; at least about 17,700,000; at least about 17,800,000; at least about 17,900,000; at least about 18,000,000; at least about 18,100,000; at least about 18,200,000; at least about 18,300,000; at least about 18,400,000; at least about 18,500,000; at least about 18,600,000; at least about 18,700,000; at least about 18,800, 000; at least about 18,900,000; at least about 19,000,000; at least about 19,100,000; at least about 19,200,000; at least about 19,300,000; at least about 19,400,000; at least about 19,500,000; at least about 19,600,000; at least about 19,700,000; at least about 19,800,000; at least about 19,900,000; at least about 20,000,000; at least about 20,100,000; at least about 20,200,000; at least about 20,300,000; at least about 20,400,000; at least about 20,500,000; at least about 20,6 00,000; at least about 20,700,000; at least about 20,800,000; at least about 20,900,000; at least about 21,000,000; at least about 21,100,000; at least about 21,200,000; at least about 21,300,000; at least about 21,400,000; at least about 21,500,000; at least about 21,600,000; at least about 21,700,000; at least about 21,800,000; at least about 21,900,000; at least about 22,000,000; at least about 22,100,000; at least about 22,200,000; at least about 22,300,000; at least about 22,400,000; at least about 22,500,000; at least about 22,600,000; at least about 22,700,000; at least about 22,800,000; at least about 22,900,000; at least about 23,000,000; at least about 23,100,000; at least about 23,200,000; at least about 23,300,000; at least about 23,400,000; at least about 23,500,000; at least about 23,600,000; at least about 23,700,000; at least about 23,800,000; at least about 23,900,000; at least about 24,000,000; at least about 24,100,000;At least about 24,200,000; at least about 24,300,000; at least about 24,400,000; at least about 24,500,000; at least about 24,600,000; at least about 24,700,000; at least about 24,800,000; at least about 24,900,000; at least about 25,000,000; at least about 25,100 ,000; at least about 25,200,000; at least about 25,300,000; at least about 25,400,000; at least about 25,500,000; at least about 25,600,000; at least about 25,700,000; at least about 25,800,000; at least about 25,900,000 or at least 26,000,000 nucleobases. ;

[0191] In some aspects, the targeting site is located at about the site of integration of SEQ ID NO: 21 or SEQ ID about 10; about 20; about 30; about 40; about 50; about 60; about 70; about 80; about 90; about 100; about 110; about 120; about 130; about 140; about 150; about 160; about 170; about 180; about 190; about 200; about 210; about 220; about 230; about 240; about 250; about 260; about 270; about 280; about 290; about 300; about 310; about 320; about 330; about 340; about 350; about 360; about 370; about 380; about 390; about 400; about 410; about 420; about 430; about 440; about 450; about 460; about 470; about 480; about 490; about 500; about 510; about 520; about 530; about 540; about 550; about 560; about 570; about 580; about 590; about 600; about 610; about 620; about 630; about 640; about 650; about 660; about 670; about 680; about 690; about 700; about 710; about 720; about 730; about 740; about 750; about 760; about 770; about 780; about 790; about 800; about 810; about 820; about 830; about 840; about 850; about 860; about 870; about 880; about 890; about 900; about 910; about 920; about 930; about 940; about 950; about 960; about 970; about 980; about 990; about 1,000; about 2,000; about 3,000; about 4,000; about 5,000; about 6,000; about 7,000; about 8,000; about 9,000; about 10,000; about 11,000; about 12,000; about 13,000; about 14,000; about 15,000; about 16,000; about 17,000; about 18,000; about 19,000; about 20,000; about 21,000; about 22,000; about 23,000; about 24,000; about 25,000; about 26,000; about 27,000; about 28,000; about 29,000; about 30,000; about 31,000; about 32,000; about 33,000; about 34,000; about 35,000; about 36,000; about 37,000; about 38,000; about 39,000; about 40,000; about 41,000; about 42,000; about 43,000; about 44,000; about 45,000; about 46,000; about 47,000; about 48,000; about 49,000; about 50,000; about 51,000; about 52,000; about 53,000; about 54,000; about 55,000; about 56,000; about 57,000; about 58,000;About 59,000; about 60,000; about 61,000; about 62,000; about 63,000; about 64,000; about 65,000; about 66,000; about 67,000; about 68,000; about 69,000; about 70,000; about 71,000; about 72,000; about 73,000; about 74, 000; about 75,000; about 76,000; about 77,000; about 78,000; about 79,000; about 80,000; about 81,000; about 82,000; about 83,000; about 84,000; about 85,000; about 86,000; about 87,000; about 88,000; about 89,000; About 90,000; about 91,000; about 92,000; about 93,000; about 94,000; about 95,000; about 96,000; about 97,000; about 98,000; about 99,000; about 100,000; about 200,000; about 300,000; about 400,000; about 500,000 ; about 600,000; about 700,000; about 800,000; about 900,000; about 1,000,000; about 1,100,000; about 1,200,000; about 1,300,000; about 1,400,000; about 1,500,000; about 1,600,000; about 1,700,000 ; about 1,800,000; about 1,900,000; about 2,000,000; about 2,100,000; about 2,200,000; about 2,300,000; about 2,400,000; about 2,500,000; about 2,600,000; about 2,700,000; about 2,800,000; about 2 ,900,000; about 3,000,000; about 3,100,000; about 3,200,000; about 3,300,000; about 3,400,000; about 3,500,000; about 3,600,000; about 3,700,000; about 3,800,000; about 3,900,000; about 4,00 0,000; about 4,100,000; about 4,200,000; about 4,300,000; about 4,400,000; about 4,500,000; about 4,600,000; about 4,700,000; about 4,800,000; about 4,900,000; about 5,000,000; about 5,100,0 00; about 5,200,000; about 5,300,000; about 5,400,000; about 5,500,000; about 5,600,000; about 5,700,000; about 5,800,000; about 5,900,000; about 6,000,000; about 6,100,000; about 6,200,000;About 6,300,000; about 6,400,000; about 6,500,000; about 6,600,000; about 6,700,000; about 6,800,000; about 6,900,000; about 7,000,000; about 7,100,000; about 7,200,000; about 7,300,000; about 7, 400,000; about 7,500,000; about 7,600,000; about 7,700,000; about 7,800,000; about 7,900,000; about 8,000,000; about 8,100,000; about 8,200,000; about 8,300,000; about 8,400,000; about 8,500 ,000; about 8,600,000; about 8,700,000; about 8,800,000; about 8,900,000; about 9,000,000; about 9,100,000; about 9,200,000; about 9,300,000; about 9,400,000; about 9,500,000; about 9,600,00 0; about 9,700,000; about 9,800,000; about 9,900,000; about 10,000,000; about 10,100,000; about 10,200,000; about 10,300,000; about 10,400,000; about 10,500,000; about 10,600,000; about 10,70 0,000; about 10,800,000; about 10,900,000; about 11,000,000; about 11,100,000; about 11,200,000; about 11,300,000; about 11,400,000; about 11,500,000; about 11,600,000; about 11,700,00 0; about 11,800,000; about 11,900,000; about 12,000,000; about 12,100,000; about 12,200,000; about 12,300,000; about 12,400,000; about 12,500,000; about 12,600,000; about 12,700,000; about 12 ,800,000; about 12,900,000; about 13,000,000; about 13,100,000; about 13,200,000; about 13,300,000; about 13,400,000; about 13,500,000; about 13,600,000; about 13,700,000; about 13,800 ,000; about 13,900,000; about 14,000,000; about 14,100,000; about 14,200,000; about 14,300,000; about 14,400,000; about 14,500,000; about 14,600,000; about 14,700,000; about 14,800,000;About 14,900,000; about 15,000,000; about 15,100,000; about 15,200,000; about 15,300,000; about 15,400,000; about 15,500,000; about 15,600,000; about 15,700,000; about 15,800,000; about 15, 900,000; about 16,000,000; about 16,100,000; about 16,200,000; about 16,300,000; about 16,400,000; about 16,500,000; about 16,600,000; about 16,700,000; about 16,800,000; about 16,900,0 00; about 17,000,000; about 17,100,000; about 17,200,000; about 17,300,000; about 17,400,000; about 17,500,000; about 17,600,000; about 17,700,000; about 17,800,000; about 17,900,000; about 18,000,000; about 18,100,000; about 18,200,000; about 18,300,000; about 18,400,000; about 18,500,000; about 18,600,000; about 18,700,000; about 18,800,000; about 18,900,000; about 19,00 0,000; about 19,100,000; about 19,200,000; about 19,300,000; about 19,400,000; about 19,500,000; about 19,600,000; about 19,700,000; about 19,800,000; about 19,900,000; about 20,000,00 0; about 20,100,000; about 20,200,000; about 20,300,000; about 20,400,000; about 20,500,000; about 20,600,000; about 20,700,000; about 20,800,000; about 20,900,000; about 21,000,000; about 21 ,100,000; about 21,200,000; about 21,300,000; about 21,400,000; about 21,500,000; about 21,600,000; about 21,700,000; about 21,800,000; about 21,900,000; about 22,000,000; about 22,100 ,000; about 22,200,000; about 22,300,000; about 22,400,000; about 22,500,000; about 22,600,000; about 22,700,000; about 22,800,000; about 22,900,000; about 23,000,000; about 23,100,000;About 23,200,000; about 23,300,000; about 23,400,000; about 23,500,000; about 23,600,000; about 23,700,000; about 23,800,000; about 23,900,000; about 24,000,000; about 24,100,000; about 24,200,000; about 24,300,000; about 24,400,000; about 24,500,000; about 24,600, 000; about 24,700,000; about 24,800,000; about 24,900,000; about 25,000,000; about 25,100,000; about 25,200,000; about 25,300,000; about 25,400,000; about 25,500,000; about 25,600,000; about 25,700,000; about 25,800,000; about 25,900,000, 26,000,000 nucleobases. ;

[0192] In some aspects, the targeted integration site is located in a high complexity sequence in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig. In some aspects, the targeted integration site comprises a high complexity sequence in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig.

[0193] In some aspects, the targeted integration site is located in a high complexity sequence in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site comprises a high complexity sequence in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig.

[0194] In some aspects, the targeted integration site is not located in a low complexity sequence in the sequence shown in SEQ ID NO:22 or in the Chr3 TI contig. In some aspects, the targeted integration site is not located in a retrotransposon sequence in the sequence shown in SEQ ID NO:22 or in the Chr3 TI contig. In some aspects, the targeted integration site is not located in an Alu repeat (CHO Alu equivalent) in the sequence shown in SEQ ID NO:22 or in the Chr3 TI contig etc. In some aspects, the targeted integration site is not located in a long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO:22 or in the Chr3 TI contig. In some aspects, the targeted integration site / locus does not contain a CHO Alu equivalent sequence.

[0195] In some aspects, the targeted integration site is not located in a low complexity sequence in the sequence shown in SEQ ID NO: 118 or in the Chr5 TI contig. In some aspects, the targeted integration site is not located in a retrotransposon sequence in the sequence shown in SEQ ID NO: 118 or in the Chr5 TI contig. In some aspects, the targeted integration site is not located in an Alu repeat (CHO Alu equivalent) or the like in the sequence shown in SEQ ID NO: 118 or in the Chr5 TI contig. In some aspects, the targeted integration site is not located in a long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO: 118 or in the Chr5 TI contig. In some aspects, the targeted integration site / locus does not contain a CHO Alu equivalent sequence.

[0196] In some aspects, the targeted integration site does not include multiple low complexity sequences in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not include a retrotransposon sequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not include an Alu repetitive sequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not include a long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs.

[0197] In some aspects, the targeted integration site does not include multiple low complexity sequences in the sequence shown in SEQ ID NO: 118 or Chr5 TI contigs. In some aspects, the targeted integration site does not include a retrotransposon sequence in the sequence shown in SEQ ID NO: 118 or Chr5 TI contigs. In some aspects, the targeted integration site does not include an Alu repeat sequence in the sequence shown in SEQ ID NO: 118 or Chr5 TI contigs. In some aspects, the targeted integration site does not include a long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO: 118 or Chr5 TI contigs.

[0198] In some aspects, the targeted integration site does not flank the low complexity sequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not flank the retrotransposon sequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not flank the Alu repetitive sequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs. In some aspects, the targeted integration site does not flank the long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs.

[0199] In some aspects, the targeted integration site is not flanked by a low complexity sequence in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is not flanked by a retrotransposon sequence in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is not flanked by an Alu repeat sequence in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is not flanked by a long interspersed nuclear element (LINE) in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig.

[0200] As used herein, the term "low complexity sequence" refers to a nucleic acid sequence characterized by the presence of repetitive sequences (also referred to as repetitive elements, repeating units, or repeats). In contrast, the term "high complexity sequence" refers to a nucleic acid sequence characterized by the absence of multiple repetitive sequences. The main types of repetitive sequences are tandem repeats and interspersed repeats, which include transposable elements (such as retrotransposons).

[0201] Retrotransposons (also referred to as class I transposable elements or transposons via RNA intermediates) are a type of genetic component that copies itself and pastes it into different genomic locations (transposons) by converting RNA back into DNA via a reverse transcription process using an RNA transposition intermediate. There are two main types of retrotransposons: long terminal repeats (LTRs) and non-long terminal repeats (non-LTRs). Retrotransposons are classified based on sequence and transposition methods. Non-LTRs mainly fall into two types: LINEs (long interspersed nuclear elements) and SINEs (short interspersed nuclear elements). Alu is the most common SINE in primates.

[0202] The Alu family is a family of repetitive elements in primate genomes (including the human genome). The Alu element is a short DNA originally characterized by the effect of the Arthrobacter luteus (Alu) restriction endonuclease. The Alu element is the most abundant transposable element, which contains more than one million copies dispersed throughout the human genome. Modern Alu elements are about 300 base pairs long, and are therefore classified as short interspersed nuclear elements (SINEs) in the repetitive DNA element category. The typical structure is 5'-part A-A5TACA6-part B-poly A tail-3' (SEQ ID NO:25), wherein part A and part B (also referred to as "left arm" and "right arm") are similar nucleotide sequences. Two major promoter "boxes" have been found in Alu: 5'A box with consensus sequence TGGCTCACGCC (SEQ ID NO:26), and 3'B box with consensus sequence GWTCGAGAC (IUPAC nucleic acid notation).

[0203] In the context of the present disclosure, references to Alu elements applied to the gray hamster sequences disclosed herein refer to CHO Alu equivalents, i.e., Alu-like elements present in the genome of gray hamsters as described in Haynes et al. (1981) Molecular and Cellular Biology 1(7):573-583. Haynes et al. described a consensus sequence of major interspersed DNA repeat sequences in the genome of Chinese hamster ovary cells (CHO cells) that is extensively homologous to human Alu sequences and mouse Bl interspersed repeat sequences. Because the CHO consensus sequence shows significant homology to the human Alu sequence, it is referred to as the CHO Alu equivalent sequence. The conserved structure around the CHO Alu equivalent family members can be identified. It is similar to the conserved structure around the human Alu and mouse Bl sequences and is represented as follows: direct repeat sequence CHO-Alu-A-rich sequence-direct repeat sequence. The consensus sequence of the CHO Alu equivalent sequence is described in Haynes et al. Figure 1 , which is incorporated herein by reference in its entirety.

[0204] Long interspersed nuclear elements (LINEs) (also called long interspersed nucleotide elements or long interspersed elements) are a group of non-LTR (long terminal repeat) retrotransposons that are widely present in the genomes of many eukaryotic organisms. They constitute approximately 21.1% of the human genome. LINEs constitute a family of transposons, each of which is about 7,000 base pairs long. The only abundant LINE in humans is LINE1. The human genome contains an estimated 100,000 truncated and 4,000 full-length LINE-1 elements. Due to the accumulation of random mutations, the sequences of many LINEs have been denatured to the point where they can no longer be transcribed or translated.

[0205] In some aspects, the targeted integration site does not comprise a CpG island in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig. In some aspects, the targeted integration site is not within a CpG island in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig. In some aspects, the targeted integration site is not flanked by a CpG island in the sequence shown in SEQ ID NO: 22 or the Chr3 TI contig.

[0206] In some aspects, the targeted integration site does not comprise a CpG island in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is not within a CpG island in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig. In some aspects, the targeted integration site is not flanked by a CpG island in the sequence shown in SEQ ID NO: 118 or the Chr5 TI contig.

[0207] CpG islands (or CG islands) are regions with a high frequency of CpG sites. Although the objective definition of a CpG island is limited, a common formal definition is a region of at least 200 bp, a GC percentage greater than 50%, and a measured to predicted CpG ratio greater than 60%. The "measured to predicted CpG ratio" can be derived when the measured value is calculated as: (number of CpGs), and the predicted value is calculated as (number of Cs * number of Gs) / length of the sequence or ((number of Cs + number of Gs) / 2) 2 / length of the sequence.

[0208] In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length and have been found in or near the promoters of approximately 40% of mammalian genes. More than 60% of human genes and nearly all housekeeping genes have their promoters embedded in CpG islands.

[0209] Based on an extensive search of the complete sequence of human chromosomes 21 and 22, DNA regions larger than 500 bp are more likely to be “true” CpG islands associated with the 5′ region of a gene if the GC content is greater than 55% and the observed to predicted CpG ratio is 65%.

[0210] CpG islands are characterized by a CpG dinucleotide content that is at least 60% of the statistically expected content (about 4%-6%), while the rest of the genome has a much lower CpG frequency (about 1%) (a phenomenon known as CG repression). Unlike CpG sites in the coding region of a gene, in most cases, CpG sites in CpG islands of promoters are non-methylated if the gene is expressed. Most methylation differences between tissues or between normal and cancer samples occur at a short distance from the CpG island (at the "CpG island shore"), rather than in the island itself.

[0211] Because histone acetylation and cytosine demethylation enhance transcription, targeted integration sites are, for example, located within loci that have above-average levels of acetylated histones and / or above-average levels of unmethylated cytosine.

[0212] Thus, in some aspects, the targeted integration site is located within, comprises, or is flanked by a subsequence of a sequence shown in SEQ ID NO:22 or Chr3 TI contigs or in SEQ ID NO:118 or Chr5 TI contigs, characterized by an above-average level of non-methylated cytosines. As used herein, an above-average level of non-methylated cytosines is considered with respect to the number of non-methylated cytosines over a certain polynucleotide length (e.g., per kilobase). Thus, the percentage of non-methylated cytosines can be calculated, for example, for the sequence shown in SEQ ID NO:22 or SEQ ID NO:118 to obtain an average level of non-methylated cytosines. Then, a subsequence in SEQ ID NO:22 or SEQ ID NO:118 can be scored according to whether the percentage of unmethylated cytosines in the subsequence (e.g., 10nt, 100nt, 1000nt, 10000nt, or more) is higher or lower than the average number of unmethylated cytosines calculated for the entire sequence shown in SEQ ID NO:22 or SEQ ID NO:118.

[0213] Similarly, in some aspects, the targeted integration site is located within, comprises, or is flanked by a subsequence in the sequence shown in SEQ ID NO:22 or Chr3 TI contigs or in SEQ ID NO:118 or Chr5 TI contigs, characterized in that it is associated with a histone having an acetylation higher than the average level. As used herein, histone acetylation higher than the average level is considered with respect to the number of acetylated histones on a certain polynucleotide length (e.g., per kilobase). Therefore, the percentage of acetylated histones can be calculated, for example, for the sequence shown in SEQ ID NO:22 or SEQ ID NO:118 to obtain the average level of acetylated histones. Then, the subsequence in SEQ ID NO:22 or SEQ ID NO:118 can be scored according to the following: the percentage of acetylated histones in the subsequence (e.g., 10nt, 100nt, 1000nt, 10000nt or more) is higher or lower than the average number of acetylated histones calculated for the entire sequence shown in SEQ ID NO:22 or SEQ ID NO:118.

[0214] For example, methods for identifying methylated markers and boundaries of transcription or open chromatin are provided in the following literature: Sharmin et al. (2016) BMC Cancer 16:88; Wang et al. (2012) Nucleic Acids Res. 40:511-29; Papin et al. (2020) J. Mol. Biol. doi:10.1016 / j.jmb.2020.09.018; Li et al. (2013) BMC Genomics 14:553; Butcher and Beck (2015) Methods 72:21-8; Chen et al. (2020) Epigenetics 22:1-22; Keller et al. (2016) Mol. Biol. Evol. 33:1019-28; Symmons et al. (2014) Genome Res. 24:390-400; or Mifsud et al. (2015) Nat. Genet. 47:598-606; Collings and Anderson (2017) Epigenetics and Chromatin 10 doi.org / 10.1186 / s13072-017-0125-5, all of which are incorporated herein by reference in their entirety.

[0215] In some aspects, the targeted integration site is located within, comprises, or is flanked by a subsequence of: a subsequence in the sequence shown in SEQ ID NO: 22 or a Chr3 TI contig, characterized by a region having an early replication initiation.

[0216] In some aspects, the targeted integration site is located within, comprises, or is flanked by a subsequence of: a subsequence in the sequence shown in SEQ ID NO: 118 or a Chr5 TI contig, characterized by a region with an early replication initiation.

[0217] Early replication initiation is associated with open chromatin and transcription regions. For example, methods for identifying replication origins and their association with chromatin states and transcription are provided in the following documents: Smith and Aladjem (2014) J. Mol. Biol. 426: 3330-41; Dellino et al. (2013) Genome Res. 23: 1-11; Boos and Ferreira (2019) Genes 10: 199; Boulos et al. (2015) FEBS Lett. 489: 2944-57; or Gomez and Brockdorff (2004) Proc. Natl. Acad. Sci. USA 101: 6923-6928; All of these documents are incorporated herein by reference in their entirety. Based on these methods, the replication origins within the sequence shown in SEQ ID NO:22 or Chr3 TI contigs or in SEQ ID NO:118 or Chr5 TI contigs can be classified and arranged as early, mid and late replication initiation regions. In some aspects, the targeted integration site is located within, comprises or is flanked by a subsequence in SEQ ID NO:22 or Chr3 TI contigs or in the sequence shown in SEQ ID NO:118 or Chr5 Ti contigs, characterized in that it is within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the replication initiation region.

[0218] In some aspects, the targeted integration site comprises the sequence set forth in SEQ ID NO:21, or a portion thereof, located at positions 20,002-20,019 of SEQ ID NO:20. In some aspects, the targeted integration site is located within or comprises a subsequence of the sequence of SEQ ID NO:21 within SEQ ID NO:20.

[0219] In some aspects, the targeted integration site comprises the sequence shown in SEQ ID NO: 117, or a portion thereof. In some aspects, the targeted integration site is located within or comprises a subsequence of the sequence of SEQ ID NO: 117 within SEQ ID NO: 116.

[0220] In some aspects, the targeted integration site is located upstream of SEQ ID NO:21 or SEQ ID NO:117.

[0221] In some aspects, the targeted integration site is located downstream of SEQ ID NO:21 or SEQ ID NO:117.

[0222] In some aspects, the targeted integration site is located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5000 about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or about 3000 nt.

[0223] In some aspects, the targeted integration site is located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5000 about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or about 3000 nt.

[0224] In some aspects, the targeted integration site is located within a sequence that is orthologous to the sequence shown in SEQ ID NO: 20, or a fragment thereof (e.g., SEQ ID NO: 21). In some aspects, the targeted integration site is located within a sequence that is paralogous to the sequence shown in SEQ ID NO: 20, or a fragment thereof (e.g., SEQ ID NO: 21).

[0225] In some aspects, the targeted integration site is located within a sequence that is orthologous to the sequence shown in SEQ ID NO: 116, or a fragment thereof (e.g., SEQ ID NO: 117). In some aspects, the targeted integration site is located within a sequence that is paralogous to the sequence shown in SEQ ID NO: 116, or a fragment thereof (e.g., SEQ ID NO: 117).

[0226] In some aspects of the disclosure, the targeted integration site is located within SEQ ID NO: 20. SEQ ID NO: 20 is a subsequence of SEQ ID NO: 22 (a 26 Mb sequence from chromosome 3 of C. cristae (Chinese hamster)) that includes 20 Kb at each site of the integration site of SEQ ID NO: 21.

[0227] In some aspects of the disclosure, the targeted integration site is located within SEQ ID NO: 116. SEQ ID NO: 116 is a subsequence of SEQ ID NO: 118 (an 18 Mb sequence from chromosome Chr5 of Cricetulus grizzly (Chinese hamster)) that includes 20 Kb at each site of the integration site of SEQ ID NO: 117.

[0228] In some aspects, the present disclosure provides isolated cells, comprising a polynucleotide sequence (exogenous nucleic acid) comprising a nucleic acid encoding a gene of interest (GOI) integrated within a specific locus in the genome of the cell, wherein the locus has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 20, SEQ ID NO: 116, or a subsequence thereof.

[0229] It should be understood that, although the sequences disclosed herein are derived from gray mice, the disclosure also encompasses orthologous sequences from other species (e.g., humans, mice, rabbits, rats, pigs, or dogs). Therefore, reference to any sequence shown in SEQ ID NOs: 14-24 and 110-120 also encompasses variant sequences with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity with those parent or reference sequences (i.e., any sequence shown in SEQ ID NOs: 14-24 and 110-120, or a fragment thereof, or a subsequence thereof), as determined via pairwise alignment using, for example, the implementation of the Needleman-Wungsch algorithm. As used herein, the term "orthologous" refers to polynucleotides with similar nucleic acid sequences separated due to speciation events, i.e., they represent homologous sequences in different organisms due to ancestral relationships, and therefore function similarly in different organisms. Therefore, sequences (or subsequences) orthologous to the sequences (or subsequences) disclosed herein are considered to be functionally equivalent, ie, capable of being used as specific loci for targeted integration equivalently to the known sequences from C. crinata disclosed herein.

[0230] In some aspects, the present disclosure provides isolated cells, comprising a polynucleotide sequence (exogenous nucleic acid) comprising a nucleic acid encoding a gene of interest (GOI) integrated within a specific locus in the genome of the cell, wherein the locus has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 20, SEQ ID NO: 116, or a subsequence thereof.

[0231] In some aspects, the length of the subsequence is about 18, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 nucleotides, wherein the subsequence comprises the sequence shown in SEQ ID NO:21 or SEQ ID NO:117.

[0232] In some aspects, the subsequence comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 nucleotides upstream of the sequence shown in SEQ ID NO:21 or SEQ ID NO:117.

[0233] In some aspects, the subsequence comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 nucleotides downstream of the sequence shown in SEQ ID NO:21 or SEQ ID NO:117.

[0234] In some aspects, the disclosure provides an isolated cell comprising a polynucleotide sequence (exogenous nucleic acid) comprising a nucleic acid encoding a gene of interest (GOI) integrated into a specific locus of the cell genome, wherein the locus (e.g., the targeted integration site of the disclosure) is a nucleotide position or a nucleotide sequence within SEQ ID NO: 20 or SEQ ID NO: 116. The disclosure also provides a method comprising introducing a polynucleotide sequence (exogenous nucleic acid) comprising a nucleic acid encoding a gene of interest (GOI) into a mammalian cell (e.g., a CHO cell), and obtaining a mammalian cell (e.g., a CHO cell), wherein the exogenous nucleic acid is integrated into a specific locus of the CHO cell genome, the locus being a nucleotide position or a nucleotide sequence within SEQ ID NO: 20 or SEQ ID NO: 116. Also provided are methods comprising (a) providing a cell comprising a polynucleotide sequence (exogenous nucleic acid) comprising a nucleic acid encoding a gene of interest (GOI), wherein the polynucleotide sequence comprises a nucleic acid encoding a gene of interest (GOI) operably linked to a promoter, and wherein the locus is a nucleotide position or nucleotide sequence within SEQ ID NO: 20 and SEQ ID NO: 116. In some aspects, the locus partially overlaps with SEQ ID NO: 20 or SEQ ID NO: 16.

[0235] In some aspects, the polynucleotide sequence comprises a nucleic acid encoding a gene of interest (GOI) integrated into a specific site at any position of SEQ ID NO: 20 or SEQ ID NO: 116.

[0236] In some aspects, the specific site is in SEQ ID 1-2000, 12001-13000, 13001-14000, 14001-15000, 15001-16000, 16001-17000, 17001-18000, 18001-19000, 19001-20000, 20001-20000, 20001-30000, 20001-40000, 20001-50000, 20001-60000, 20001-70000, 20001-80000, 20001-90000, 20001-10000 0001-21000、21001-22000、22001-23000、23001-24000、24001-25000、25001-26000、26001-27000、27001-28000、28001-29000、29001-30000、30001- 31000, 31001-32000, 32001-33000, 33001-34000, 34001-35000, 35001-36000, 36001-37000, 37001-38000, 38001-39000, 39001-40000 or 40001-40020.

[0237] In some aspects, the specific site is at a position within SEQ ID NO: 116 selected from nucleotides spanning the following position numbers: 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001-11000, 11001-12000, 12001-13000, 13001-14000, 14001-15000, 15001-16000, 16001-17000, 17001-18000, 18001-19000, 19001-2000 0000, 20001-21000, 21001-22000, 22001-23000, 23001-24000, 24001-25000, 25001-26000, 26001-27000, 27001-28000, 28001-29000, 29001-3000 0, 30001-31000, 31001-32000, 32001-33000, 33001-34000, 34001-35000, 35001-36000, 36001-37000, 37001-38000, 38001-39000, 39001-40000, 4 0001-41000、410001-42000、42001-43000、43001-44000、44001-45000、45001-46000、46001-47000、47001-48000、49001-50000、50001-51000、51 001-52000、52001-53000、53001-54000、54001-55000、55001-56000、56001-57000、57001-58000、58001-59000、59001-60000、60001-61000、61001 -62000, 62001-63000, 63001-64000, 64001-65000, 65001-66000, 66001-67000, 67001-68000, 68001-69000, 69001-70000, 70001-71000, 71001-72 000、72001-7300、73001-74000、74001-75000、75001-76000、76001-77000、77001-78000、78001-79000、79001-80000、80001-81000、81001-82000、82001-83000、83001-84000、84001-85000、85001-86000、86001-87000、87001-88000、88001-89000、89001-90000、90001-91000、91001-92000、92001-93000、93001-94000、94001-95000、95001-96000、96001-97000、97001-98000、98001-99000、99001-100000、100001-101000、101001-102000、102001-103000、103001-104000、104001-105000、105001-106000、106001-107000、107001-108000、108001-109000、109001-110000、110001-111000、111001-112000、112001-113000、113001-114000、114001-115000、115001-116000、116001-117000、117001-118000、118001-119000、119001-120000、120001-121000、121001-122000、122001-123000、123001-124000、124001-125000、125001-126000、126001-127000、127001-128000、128001-129000、129001-130000、130001-131000、131001-132000、132001-133000、133001-134000、134001-135000、135001-136000、136001-137000、137001-138000、138001-139000、139001-140000、140001-141000、141001-142000、142001-143000、143001-144000、144001-145000、145001-146000、146001-147000、147001-148000、148001-149000、149001-150000、150001-151000、151001-152000、152001-153000、153001-154000、154001-155000、155001-156000、156001-157000, 157001-158000, 158001-159000, 159001-160000, 160001-161000, 161001-162000, 162001-163000, 163001-164000, 164001-165000, 165001-166000, 166001-167000, 167001-168000, 168001-169000, 169001-170000, 170001- 171000, 171001-172000, 172001-173000, 173001-174000, 174001-175000, 175001-176000, 176001-177000, 177001-178000, 178001-179000, 179001-180000, 180001-181000, 181001-182000, 182001-183000, 183001-184000 or 184001-185000.

[0238] In some aspects, the specific site is in SEQ ID At a position within NO: 20, the position is selected from nucleotides spanning the following position numbers: 19000-21000, 18000-22000, 17000-23000, 16000-24000, 15000-25000, 14000-26000, 13000-27000, 12000-28000, 11000-29000, 10000-30000, 9000-31000, 8000-32000, 7000-33000, 6000-34000, 5000-35000, 4000-36000, 3000-37000, 2000-38000, 1000-39000, or 1-40020.

[0239] In some aspects, the specific site is at a position within SEQ ID NO: 20 selected from nucleotides spanning the following position numbers: 19000-19100, 19100-19200, 19200-19300, 19300-19400, 19400-19500, 19500-19600, 19600-19700, 19700-19800, 19800-199 00, 19900-20000, 20000-20100, 20100-20200, 20200-20300, 20300-20400, 20400-20500, 20500-20600, 20600-20700, 20700-20800, 20800-20900 or 20900-21000.

[0240] In some aspects, the polynucleotide sequence comprises a nucleic acid encoding a gene of interest (GOI) integrated into a specific site at any position of SEQ ID NO: 20, wherein the specific site is at the following position within SEQ ID NO: 20, the position consisting of nucleotides spanning the following position numbers: 20000-20020, 19990-20030, 19980-20040, 19970-20050, 19960-20060, 19950-20070, 19940-20080, 19930-20090, 19920-20100, 19910-20110, 19900-20120. 120, 19890-20120, 19880-20130, 19870-20140, 19860-20150, 19850-20160, 19840-20170, 19830-20180, 19820-20190, 19810-20200, 19800-20210, 19790-20220, 19780-20230, 19770-20230, 1 9760-20240, 19750-20250, 19740-20260, 19730-20270, 19720-20280, 19710-20290, 19700-20300, 19690-20310, 19680-20320, 19670-20330, 19660-20340, 19650-20350, 19640-20360, 19630- 20370, 19620-20380, 19610-20390, 19600-20400, 19590-20410, 19580-20420, 19570-20430, 19560-20440, 19550-20450, 19540-20460, 19530-20470, 19520-20480, 19510-20490 or 19500-20500.

[0241] In some aspects, the polynucleotide sequence comprises a nucleic acid encoding a gene of interest (GOI) integrated into a specific site (hotspot) within SEQ ID NO: 20 (a genomic sequence comprising hotspot 1) or SEQ ID NO: 116 (a genomic sequence comprising hotspot 2) or any position within partially overlapping SEQ ID NO: 20 or SEQ ID NO: 116.

[0242] In some aspects, the specific site at a position within SEQ ID NO:20 is selected from nucleotide positions or subsequences spanning position numbers 20,002-20,019 (corresponding to the 18-mer sequence set forth in SEQ ID NO:21).

[0243] In some aspects, the specific site is at a position within SEQ ID NO: 20 selected from the group consisting of nucleotide positions 19900, 19901, 19902, 19903, 19904, 19905, 19906, 19907, 19908, 19909, 19910, 19911, 19912, 19913, 19914, 19915, 19916, 19917, 19918, 19919, 19920, 19921, 19922, 19923, 19924, 19925, 19926, 19927, 19928, 19929, 19920, 19921, 19922, 19923, 19924, 19925, 19926, 19927, 19928, 26. 19927, 19928, 19929, 19930, 19931, 19932, 19933, 19934, 19935, 19936, 19937, 19938, 19939, 19949, 19941, 19942, 19943, 19944, 19945, 19946 , 19947, 19948, 19949, 19950, 19951, 19952, 19953, 19954, 19955, 19956, 19956, 19958, 19959, 19960, 19961, 19962, 19963, 19964, 19965, 19966, 19 967, 19968, 19969, 19970, 19971, 19972, 19973, 19974, 19975, 19976, 19977, 19978, 19979, 19980, 19981, 19982, 19983, 19984, 19985, 1998 6. 19987, 19988, 19989, 19990, 19991, 19992, 19993, 19994, 19995, 19996, 19997, 19998, 19999, 20000, 20001, 20002, 20003, 20004, 20005, 20006, 2 20007, 20008, 20009, 20010, 20011, 20012, 20013, 20014, 20015, 20016, 20017, 20018, 20019, 20020, 20021, 20022, 20023, 20024, 20025, 20026, 200 27, 20028, 20029, 20030, 20031, 20032, 20033, 20034, 20035, 20036, 20037, 20038, 20039, 20040, 20041, 20042, 20043, 20044, 20045, 20046, 20047,20048, 20049, 20050, 20051, 20052, 20053, 20054, 20055, 20056, 20057, 20058, 20059, 20060, 20061, 20062, 20063, 20064, 20065, 20066, 20067, 20068, 20069, 20070, 20071, 20072, 20073, 200 74, 20075, 20076, 20077, 20078, 20079, 20080, 20081, 20082, 20083, 20084, 20085, 20086, 20087, 20088, 20089, 20090, 20091, 20092, 20093, 20094, 20095, 20096, 20097, 20098, 20099 or 20100.

[0244] The present disclosure also provides methods that allow the generation of landing pad cell lines and expression cell lines without any prior knowledge of the genomic sequences surrounding the parental plasmid and the identification of additional hot spots in the genome of the parental cell line. The general TI technology utilizes one or more site-specific endonucleases that target parental plasmid sequences in the parental cell line that are not present in the landing pad plasmid. The advantage of this strategy is that no knowledge of the flanking genomic DNA sequences is required. For example, Figure 4A The genomic sequence targeted by CRISPR / Cas is shown, indicated by a solid box next to the scissors representing CRISPR / Cas. Fig. 8A and Figure 8B It is shown that the sequence targeted by CRISPR / Cas is internal to the parental plasmid. Boxes with vertical and wavy lines indicate regions of homology between different plasmids.

[0245] According to these new strategies, for example Figure 2 As shown in and related disclosures, a parental cell line with a high expression titer (e.g., 3-4 g / L for an antibody) and a low copy number (e.g., 2) will first be selected. Once such a cell line (i.e., a "hot cell line") is identified, the hot cell line can be used according to two different strategies. In both strategies, the landing pad plasmid encodes a marker (e.g., a fluorescent marker such as blmCherry) and expresses a selection marker (e.g., puromycin resistance) that is different from the parent plasmid present in the parental cell line, and the polynucleotide sequence encoding the marker is flanked by heterologous site-specific recombination sites (SSRS). Fig. 12A , Fig. 12B , Fig.13 and Fig.14The exemplary SSRS shown in are two Lox sites (LoxP and Lox511), which are targets of Cre recombinase. However, alternative SSRS (e.g., Lox, Frt, att, or a combination thereof) can be used to practice these strategies as disclosed below. For example, the Lox and Frt combination is depicted in Fig.15 and for example in Fig.19 The use of att sites (attachment sites) is shown in FIG.

[0246] In the presence of a site-specific nuclease (e.g., CRISPR / Cas) and a landing pad plasmid, the first GOI (e.g., mAb expression cassette) in the parental cell line is either replaced with a landing pad displaying mCherry flanked by Lox sites (Strategy A), or is deleted and the landing pad plasmid integrated into an alternative locus in the genome of the hot cell line (Strategy B). Thus, in Strategy A, the landing pad plasmid is inserted into a hotspot that supports high expression, which will be the same hotspot used in the parental cell line. In Strategy B, the first GOI (e.g., mAb expression cassette) in the parental cell line will be removed and the landing pad plasmid will be inserted at an alternative position in the genome of the parental cell line. Since the parental cell line is a hot cell, identification of additional hotspots will result in a landing pad cell line capable of generating an expression cell line with preferred attributes (e.g., high titer). See. Fig. 8A .

[0247] The present disclosure provides a method for identifying a landing pad cell line, the method comprising: (1) removing the first GOI from a plasmid integrated into the genomic sequence of a parent cell (e.g., a thermocyte), thereby generating a parent cell population without the first GOI; (2) integrating a landing pad plasmid comprising at least one marker (e.g., Cherry) at an alternative genomic locus in the parental cell population of (1), thereby generating a library or candidate cells; and (3) screening the library of candidate cells comprising at least one copy of the landing pad plasmid integrated at at least one alternative genomic locus, wherein the candidate cell line is selected if the candidate cell line meets the following desired attributes: (a) cell titer is above a predetermined threshold level; (b) plasmid copy number is at a predetermined value; (c) RNA expression level is above a predetermined threshold level; or (d) multiple plasmid copies, if present, have a specific plasmid configuration.

[0248] In some aspects, only cells containing a landing pad plasmid in a newly identified hotspot are selected. In some aspects, cells containing more than one landing pad plasmid in a newly identified hotspot are selected. In some aspects, the parent cell is a historical cell line (e.g., a cell line characterized by high expression titers of a GOI (e.g., an antibody or antigen-binding portion thereof)). In some aspects, the library of candidate cells is a library generated by randomly integrating a landing pad sequence at multiple locations in the genome of a parent cell, wherein the parent cell is modified, for example, by deletion / excision / removal of a protein encoding a target protein (e.g., an antibody or antigen-binding portion thereof). In some aspects, the method selects hot cells having at least one landing pad plasmid integrated in a new hotspot. In some aspects, the parent cell line is a CHO cell line.

[0249] The present disclosure provides a method of generating landing pad cells, the method comprising integrating a landing pad plasmid into the genome of a parent cell (e.g., a CHO thermocell) at a targeted integration site using homologous recombination (using CRISPR / Cas), wherein the sequence targeted for homologous recombination is located in the parent plasmid, i.e., the sequence targeted for homologous recombination is not a genomic sequence, wherein the homologous recombination site of the landing pad plasmid recombines with the corresponding homologous recombination site of the parent plasmid, thereby integrating the landing pad plasmid at an internal position within the parent plasmid inserted into the genomic DNA of the parent cell.

[0250] In some aspects, each landing pad plasmid comprises (i) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (ii) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (i); and (iii) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (ii), which are homologous to corresponding homologous recombination sites in the parental plasmid.

[0251] The present disclosure also provides a method of generating an expression cell, the method comprising integrating a GOI plasmid (e.g., a plasmid encoding an antibody or antigen-binding portion thereof) into the genome of a landing pad cell (e.g., a CHO thermocell) disclosed above using site-specific recombinase recombination (e.g., using a Cre / Lox system), wherein the site-specific recombination site of the landing pad plasmid is recombined with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI. In some aspects, the resulting expression plasmid comprises (i) a polynucleotide sequence comprising a nucleic acid encoding at least one GOI; and (ii) two SSRS flanking the polynucleotide of (i).

[0252] Also provided is a method for generating an expression cell, the method comprising: (a) integrating a landing pad plasmid into the genome of a parent cell (e.g., a parent thermocell) at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is located in the parent plasmid, and wherein the homologous recombination site in the landing pad plasmid recombines with a corresponding homologous recombination site of the parent plasmid in a landing pad at a different genomic locus, thereby integrating the landing pad plasmid at an internal position within the landing pad at a different genomic locus in the genomic DNA of the parent cell; and (b) integrating a GOI plasmid into the genome of the landing pad cell using a site-specific recombinase recombination, wherein the site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell. In some aspects of the method, each landing pad plasmid comprises (i) at least one polynucleotide sequence comprising a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (ii) two SSRS flanking the polynucleotide sequence of (i); and (iii) two homologous recombination sites located at the 5' and 3' ends of the SSRS of (ii), which are homologous to the corresponding homologous recombination sites in the parent plasmid. In some aspects of the method, the resulting expression plasmid comprises (i) a polynucleotide sequence comprising a nucleic acid encoding at least one GOI; and (ii) two SSRS flanking the polynucleotide of (i).

[0253] Also provided is a method for generating a landing pad cell, the method comprising: (a) removing a parent plasmid or a portion thereof from a first hotspot location in a parent cell line; and (b) integrating the landing pad plasmid into a second hotspot location in the genome of the parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is present in the parent plasmid, and wherein the homologous recombination site of the landing pad plasmid recombines with the corresponding homologous recombination site of the parent plasmid, thereby integrating the landing pad plasmid at an internal location within the parent plasmid inserted into the genomic DNA of the parent cell. In some aspects of the method, the landing pad plasmid comprises (i) a polynucleotide sequence containing a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (ii) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (i); and (iii) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (ii), which are homologous to the corresponding homologous recombination sites in the parent plasmid. Step (a) will generate a cell population derived from a parental cell line (e.g., a hot cell line) without a first GOI (e.g., an antibody with high expression levels in the parental cell line). In step (b), inserting a landing pad plasmid into the genome of the cell population of step (a) will generate a cell population that will contain integrated landing cell pads at multiple locations, so that the cell population can be screened to identify new hot cells and their corresponding hot spots.

[0254] Also provided is a method of generating an expressing cell, the method comprising: (a) removing a parental plasmid from a first hotspot location in a parental cell line, (b) integrating a landing pad plasmid into a second hotspot location in the genome of the parental cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is present in the parental plasmid, wherein each landing pad plasmid comprises, for example, (i) a polynucleotide sequence comprising a nucleic acid encoding at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker; (ii) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (i); and (iii) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (ii), wherein homologous to corresponding homologous recombination sites in the parent plasmid; and wherein the homologous recombination sites of the landing pad plasmid recombine with corresponding homologous recombination sites of the parent plasmid, thereby integrating the landing pad plasmid at an internal position within the parent plasmid inserted into the genomic DNA of the parent cell, and (c) integrating the GOI plasmid into the genome of the landing pad cell using a site-specific recombinase recombination, wherein the expression plasmid comprises, for example, (i) a polynucleotide sequence containing a nucleic acid encoding a GOI; and (ii) two SSRs flanking the polynucleotide of (1); wherein the site-specific recombination sites of the landing pad plasmid recombine with corresponding site-specific recombination sites of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

[0255] In some aspects, the landing pad cells comprise plasmids having a topology corresponding to the following description: CG / -[P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1]- / CG; CG / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG; CG / -[P1]-([P2]-[SSRS]-[M]-[P2])n-[P1]- / CG; CG / -([P2]-[SSRS]-[M]-[P2])n- / CG, CG / -[P1]-([P2]-[M]-[SSRS]-[P2])n-[P1]- / CG; or CG / -([P2]-[M]-[SSRS]-[P2])n- / CG wherein CG is a parental cell genomic sequence flanking the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one marker; [SSRS] is a site-specific recombination site (SSRS), and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10.

[0256] Note that the labels [P1], [P2], and [SSRS] in any formula in this disclosure are merely descriptors of the origin or type of components used to represent the topology of the construct. The nucleic acid sequences of each [P1] and [P2] component are different, i.e., the nucleic acid sequence of the first [P1] is different from the nucleic acid sequence of the second [P1], but they have a common origin (i.e., the parent plasmid). Similarly, the nucleic acid sequence of the first [P2] is different from the nucleic acid sequence of the second [P2], but they have a common origin (i.e., the landing pad plasmid). In some aspects, the CG sequence in the landing pad cell is different from the CG sequence in the parental cell line, i.e., the plasmid is located in a hotspot that is different from the original hotspot in the parental cell line.

[0257] [SSRS] components are, for example, Cre / Lox sites, and each of them may have different sequences. However, in some aspects, in any formula present throughout the disclosure that includes a [SSRS] pair, one of the [SSRS] shown is optional. When integrating using, for example, a serine integrase, a single [SSRS] is required. Therefore, in those specific aspects, a single att site (e.g., attP site) may be present, rather than a [SSRS] pair.

[0258] In some aspects, the topology of the plasmid integrated into the expression cell corresponds to the following description: CG / -[P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1]- / CG; CG / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG; CG / -[P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1]- / CG; CG / -([P2]-[SSRS]-[P3]-[P2])n- / CG; CG / -[P1]-([P2]-[P3]-[SSRS]-[P2])n-[P1]- / CG; or CG / -([P2]-[P3]-[SSRS]-[P2])n- / CG, wherein CG is a parental cell genomic sequence that flanks the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid comprising a gene of interest (GOI); [SSRS] is a site-specific recombination site (SSRS); and n is an integer between 1 and 10. In some aspects, n is 1. In some aspects, n is 2. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6. In some aspects, n is 7. In some aspects, n is 8. In some aspects, n is 9. In some aspects, n is 10. In some aspects, the CG sequence in the landing pad cell is different from the CG sequence in the parental cell line, i.e., the plasmid is located in a hotspot that is different from the original hotspot in the parental cell line.

[0259] In some aspects, homologous recombination is mediated by the CRISPR / Cas system, TALEN system or ZFN system described in detail below. In some aspects, the homologous recombination system (e.g., CRISPR / Cas system) further comprises a single guide RNA (sgRNA). Depending on the homologous recombination used, additional components may be required, as disclosed in detail below.

[0260] In some aspects, site-specific recombinase recombination site (SSRS) is a Tyr recombinase site, a Tyr integrase site, a serine resolvase / convertase site, a serine integrase site or a combination thereof. In some aspects, the Tyr recombinase site includes a Cre, Dre, Flp, KD, B3 or B3 Tyr recombinase site. In some aspects, the Tyr integrase site includes a λ (lambda), HK022 or HP1 Tyr integrase site. In some aspects, the serine resolvase / convertase site includes a γδ (gamma delta), ParA, Tn3 or Gin serine resolvase / integrase site. In some aspects, the serine integrase site includes a PhiC31, Bxb1, pr R4 serine integrase site. In some aspects, the Tyr recombinase site includes a Cre Tyr recombinase site. In some aspects, SSRS is a LoxP site. In some aspects, the LoxP site includes the nucleic acid sequence shown in SEQ ID NO:1 (wild-type LoxP). In some aspects, the LoxP site includes a mutant LoxP site. In some aspects, the mutant LoxP site includes the nucleic acid sequence shown in SEQ ID NO:2 (mutant LoxP). In some aspects, the mutant LoxP site includes a nucleic acid selected from, for example, the following: SEQ ID NO:3 (Lox 511); SEQ ID NO:4 (Lox 5171); SEQ ID NO:5 (Lox2272); SEQ ID NO:6 (Lox M2); SEQ ID NO:7 (Lox M3); SEQ ID NO:8 (Lox M7); SEQ ID NO:9 (Lox M11); SEQ ID NO:10 (Lox 71); and SEQ ID NO:11 (Lox 66). In some aspects, the Tyr recombinase site includes a Flp Tyr recombinase site. In some aspects, SSRS is a short flip enzyme recognition target (FRT) site. In some aspects, the serine integrase site includes an att site (e.g., attP or attB site).

[0261] In some aspects, each SSRS in a pair of SSRS in the plasmid disclosed herein can belong to different categories. For example, the first SSRS can be, for example, a Tyr recombinase site, and the second SSRS can be, for example, a Ser integrase site. In some aspects, SSRS is to including two sites selected from the following: wild-type LoxP, mutant LoxP site, Lox 511 site, Lox5171 site, Lox 2272 site, Lox M2 site, Lox M3 site, Lox M7 site, Lox M11 site, Lox 71 site, Lox 66 site or any combination thereof. In some aspects, SSRS is to including Lox P site and Lox511 site. In some aspects, SSRS is to including Lox P site and Frt site. In some aspects, SSRS is to including two aat sites, for example, two attP sites. In some aspects, SSRS is to including two aat sites, for example, two attR sites. In some aspects, SSRS is to including Lox 2272 site and Lox M3 site. In some aspects, the SSRS pair includes a Lox m3 site and a Lox m7 site.

[0262] In some aspects, the plasmid disclosed herein comprises at least one single selection marker. In some aspects, the plasmid disclosed herein comprises a single selection marker. In some aspects, the plasmid disclosed herein comprises more than one single selection marker (e.g., two selection markers). In some aspects, at least one selection marker is glutamine synthetase (GS). In some aspects, at least one selection marker is dihydrofolate reductase (DHFR). In some aspects, at least one selection marker includes glutamine synthetase (GS) marker and dihydrofolate reductase (DHFR) marker. There are several selection markers suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells to obtain high productivity. See Yeo et al. (2017) Biotechnol J 12 (12), which is incorporated herein by reference in its entirety.

[0263] In some aspects, at least one selection marker is a drug resistance gene (e.g., an antibiotic resistance gene). In some aspects, the antibiotic resistance gene is selected from actinomycin D resistance gene, bleomycin resistance gene, chloramphenicol resistance gene, G418 resistance gene, hygromycin resistance gene, mitomycin C resistance gene, mycophenolic acid resistance gene, puromycin resistance gene and any combination thereof. In some aspects, the antibiotic resistance gene is a puromycin resistance gene. In some aspects, the puromycin resistance gene is puromycin-N-acetyltransferase.

[0264] In some aspects, at least one detectable marker includes a protein (e.g., a fluorescent protein). In some aspects, the fluorescent protein is mCherry. In some aspects, the fluorescent protein is selected from GFP, ZsGreen1, AcGFP1, EGFP, GFPuv, AcGFP, EBFP, EYFP, ECFP, tdTomato, mCherry, DsRed, AmCyan, ZsGreen, ZsYellow, DsRed2, DsRed-Express, HcRed, AsRed, mOrange, mOrange2, mPlum, mStrawberry, mBanana, YFP, mRaspberry, HcRed1, E2-Crimson, and any combination thereof.

[0265] In some aspects, the parent cell is selected from Chinese hamster ovary (CHO) cells, HEK293 cells and NS0 cells or derivatives or equivalents thereof. In some aspects, the CHO cell is a CHO DG44 cell or a CHO K1 cell.

[0266] In some aspects, GOI encodes at least one polypeptide (e.g., an antibody or fusion protein). In some aspects, the antibody specifically binds to T cell immunoglobulin and mucin domain protein 3 (TIM3), tau protein (e.g., N-terminal fragment of tau (eTau)), or immune checkpoint protein (e.g., PD-1 or PD-L1). In some aspects, the antibody is nivolumab. In some aspects, GOI is the heavy chain (HC) of the antibody. In some aspects, GOI is the light chain (LC) of the antibody. In some aspects, GOI comprises HC and LC of the antibody (e.g., a bicistronic construct). In some aspects, GOI is a bispecific antibody or a portion thereof (e.g., HC or LC of a bispecific antibody or any combination thereof). In some aspects, the expression plasmid comprises one, two, or more than two copies of GOI.

[0267] In some aspects, the methods disclosed herein include determining the expression of a GOI or a marker disclosed herein. In some aspects, the expression of a GOI or a marker is determined quantitatively and / or qualitatively. In some aspects, the expression of a GOI or a marker is determined, for example, by cell sorting, FACS, cell surface staining, Western blot, Northern blot, column chromatography, capillary electrophoresis, microfluidics, UV absorbance, immunohistochemistry, cell size, secreted protein levels, transcript levels, or any combination thereof.

[0268] In some aspects, the landing pad plasmid (second GOI plasmid) or expression plasmid (P4) is integrated into the genome of the cell at a copy number of 1. In some aspects, the landing pad plasmid (second GOI plasmid) or expression plasmid (P4) is integrated into the genome of the cell at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0269] In some aspects, the 5' homologous recombination site of the plasmid disclosed herein comprises the polynucleotide sequence of SEQ ID NO: 18, or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 19, or a subsequence thereof.

[0270] In some aspects, the 5' homologous recombination site of the plasmid disclosed herein comprises the polynucleotide sequence of SEQ ID NO: 114, or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 115, or a subsequence thereof.

[0271] In some aspects, an isolated cell or a population of isolated cells of the present disclosure comprises a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI) integrated within a specific locus of the cell genome, wherein the locus comprises a nucleotide subsequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 116. In some aspects, the methods disclosed herein comprise introducing a polynucleotide sequence comprising a nucleic acid encoding at least one gene of interest (GOI) into a cell (e.g., a CHO cell or another suitable cell line) and obtaining a cell (e.g., a CHO cell), wherein the exogenous nucleic acid is integrated into a specific locus of the cell (e.g., CHO cell) genome, wherein the locus comprises a nucleotide subsequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 116. In some aspects, the methods disclosed herein comprise (a) providing a cell comprising a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), wherein the polynucleotide sequence comprises a nucleic acid encoding a gene of interest (GOI) operably linked to a promoter, and wherein the locus comprises a nucleotide subsequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 116. In some aspects, a nucleotide subsequence selected from SEQ ID NO:20 comprises the sequence shown in SEQ ID NO:21. In some aspects, a nucleotide subsequence selected from SEQ ID NO:116 comprises the sequence shown in SEQ ID NO:117. In some aspects, a nucleotide subsequence selected from SEQ ID NO:20 consists of the sequence shown in SEQ ID NO:21. In some aspects, a nucleotide subsequence selected from SEQ ID NO:116 consists of the sequence shown in SEQ ID NO:117. In some aspects, a nucleotide subsequence selected from SEQ ID NO:20 is a subsequence upstream (towards the 5' end of SEQ ID NO:20) of the sequence shown in SEQ ID NO:21. In some aspects, a nucleotide subsequence selected from SEQ ID NO:20 is a subsequence downstream (towards the 3' end of SEQ ID NO:20) of the sequence shown in SEQ ID NO:21. In some aspects, a nucleotide subsequence selected from SEQ ID NO: 116 is a subsequence upstream (towards the 5' end of SEQ ID NO: 116) of the sequence shown in SEQ ID NO: 117. In some aspects, a nucleotide subsequence selected from SEQ ID NO: 116 is a subsequence downstream (towards the 3' end of SEQ ID NO: 116) of the sequence shown in SEQ ID NO: 117.

[0272] The present disclosure provides landing pad cell lines containing a single landing pad plasmid. However, landing pad cell lines with more than one landing pad plasmid provide the opportunity to further improve the expression of multi-subunit biologics, such as bispecific monoclonal antibodies (mAbs). Therefore, the cell screening methods disclosed herein can be used to identify landing pad cell lines with two landing pad plasmids in the same locus (i.e., dual landing pad cells). This ensures equal expression from both landing pad plasmids because they are present in the same genomic locus.

[0273] The dual landing pads of the present disclosure can integrate in four different orientations (head-to-head, tail-to-tail, tail-to-head, and head-to-tail). When using a single site-directed recombinase (such as Cre / Lox or Flp / Frt), the head-to-head and tail-to-tail configurations are often used because they are functionally indistinguishable from each other. Unlike the tail-to-head and head-to-tail configurations, which can result in the deletion of one of the landing pads in the presence of Cre / Lox, the head-to-head and tail-to-tail configurations simply undergo inversion, resulting in the same starting configuration.

[0274] When the second GOI plasmid is used with each of the four dual landing pad configurations (head-to-head, tail-to-tail, tail-to-head, and head-to-tail), the head-to-head and tail-to-tail configurations can each generate two cell lines, where the sequence between the two recombination sites flanking the plasmid junction can be inverted, otherwise the two cell lines are identical. When the head-to-tail or tail-to-head configuration is used with the second GOI plasmid, a cell line with two second GOI plasmids is generated. However, if there is a sufficient amount of Cre activity, one of the second GOI plasmids can be removed, thereby generating a second GOI plasmid cell line with a single second GOI plasmid.

[0275] If the landing pad uses the Frt recognition site of Flp instead of the Lox site (e.g., Lox 511), and both Cre / Lox and Flp are used, the same outcome will result, i.e., deletion in the tail-to-head and head-to-tail orientations, while the head-to-head and tail-to-tail orientations undergo inversions. However, in the tail-to-tail and head-to-head configurations, the recombination of the second GOI plasmid into the double landing pad using attP / attB with integrase does not result in inversions, but in the tail-to-head and head-to-tail configurations, deletion of one of the landing pads can still occur. If each landing pad has a single attP site, a single integration of the second GOI plasmid with a single attB site will occur, resulting in no deletion in any of the four double landing pad configurations.

[0276] As used herein, the term "single landing pad" refers to a landing pad comprising a single landing pad plasmid or a second GOI plasmid. As used herein, the term "dual landing pad" refers to a landing pad comprising two landing pad plasmids or a second GOI plasmid.

[0277] The use of dual landing pads provides an alternative method for producing biologics comprising different GOIs (e.g., antibodies comprising heavy and light chains). In one aspect, the disclosure provides methods and compositions wherein the second GOI plasmid comprises multiple expression cassettes encoding, for example, the heavy and light chains of an antibody. In another aspect, each expression cassette can be in a different second GOI plasmid, and both second GOI plasmids will be located in the dual landing pad.

[0278] The use of dual landing pad cell lines has advantages over landing pad cell lines with a single landing pad (i.e., a landing pad containing a single second GOI plasmid). In the case of a single landing pad cell line, all expression cassettes required for the preparation of a multi-component biologic must be placed in a single second GOI plasmid because the cell line only accommodates a single second GOI. This is not the case with dual landing pad cell lines. Dual landing pad cell lines provide the opportunity to design with a greater level of expression diversity, thereby providing the opportunity to produce an expression cell line with superior characteristics. Diversity can be generated in a variety of ways using second GOI plasmids of different configurations. In one case, the second GOI plasmid contains all expression cassettes required for the preparation of a composite biologic in a unique configuration. In a second case, the second GOI plasmid may contain a subset of expression cassettes that need to be present in the same cell to prepare an expression cell line. In a third case, this case is a combination of the two preceding cases, wherein one or more second GOI plasmids have all expression cassettes required for the preparation of a composite biologic in a unique configuration, and a set of second GOI plasmids contains a subset of all expression cassettes in one or more unique configurations.

[0279] It should be understood that the same methods disclosed herein for generating dual landing pads can be used to generate cell lines with higher order combinations of landing pad plasmids. For example, the methods disclosed herein for identifying landing pad cell lines with two landing pad plasmids in a hotspot can be used to select landing pad cell lines with three, four or more landing pad plasmids. Landing pad cell lines and expression cells with hotspots containing more than two landing pad plasmids can be used, for example, to produce biologics containing more than two different subunits.

[0280] Although in some aspects, the dual landing pad configuration can contain two landing pad plasmids with the same recombinase or Int recognition sequence, each landing pad plasmid can be made to have a unique recombination "address" (i.e., each landing pad plasmid becomes addressable). In the case of recombinases such as Cre and Flp, four unique recognition sequences can be used. Therefore, each landing pad plasmid will have a unique recognition site pairing. In some aspects, four incompatible Lox sites can be used. See Langer, SJ, Ghafoori, AP, Byrd, M. and Leinwand, L. (2002) A genetic screenidentifies novel non-compatible loxP sites. Nucleic Acids Res., 30, 3067-3077; Missirlis, PI, Smailus, DE and Holt, RA (2006) A high-throughput screenidentifying sequence and promiscuity characteristics of the loxP spacerregion in Cre-mediated recombination. BMC Genomics, 7, 73.; and Siegel, RW, Jain, R., and Bradbury, A. (2001) Using an in vivo phagemid system to identify non-compatible loxP sequences. FEBS Lett., 505, 467-473.

[0281] Examples of additional strategies include replacing two Lox sites with two incompatible Frt sites and using Cre with Frt (see Lauth, M., Spreafico, F., Dethleffsen, K., and Meyer, M. (2002) Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases. Nucleic Acids Res., 30, e115), using integrase with two to four incompatible aat sites (see Jusiak, B., Jagtap, K., Gaidukov, L., Duportet, X., Bandara, K., Chu, J., Zhang, L., Weiss, R., and Lu, TK (2019) Comparison of IntegrasesIdentifies Bxb1-GA Mutant as the Most Efficient Site-Specific Integrase System in Mammalian Cells. ACS Synth Biol, 8, 16-24), using more than one integrase, such as the integrases of BxB1 and phiC3 (see Smith, MC, Brown, WR, McEwan, AR and Rowley, PA (2010) Site-specific recombination by phiC31 integrase and other large serinerecombinases. Biochem. Soc. Trans., 38, 388-394), and combinations thereof. Using a single att site in each landing pad is sufficient to insert the second GOI plasmid into each landing pad. In this case, the second GOI plasmid needs to be circular, as the linear plasmid will effectively restrict the chromosome. It is also clear that the landing pad can contain multiple att sites, so that each contains a unique address.

[0282] A dual landing pad configuration in which the landing pads have unique addresses can also be used to generate expression cell lines with a more defined diversity than when the landing pads are not addressable, as well as generating higher diversity than landing pad cell lines with a single landing pad.

[0283] Another application of addressable landing pads is the option to express two independent biological products, each with its own independent function. One of the biological products can help the expressing cell line express the second biological product, or the first biological product can cause a specific post-translational modification of the second biological product or modify some other component of the expressing cell line.

[0284] In some aspects, the methods, cells, cell lines, or kits disclosed herein comprise at least two landing pad plasmids or at least two expression plasmids in series. In other words, in some aspects, the following formula CG / -([P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1])- / CG; CG / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG; CG / -([P1]-([P2]-[SSRS]-[M]-[P2])n-[P1])- / CG; CG / -([P1]-([P2]-[M]-[SSRS]-[P2])n-[P1])- / CG; CG / -([P2]-[SSRS]-[M]-[P2])n- / CG; CG / -([P2]-[M]-[SSRS]-[P2])n- / CG; CG / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG; CG / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG; CG / -([P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1])- / CG; CG / -([P1]-([P2]-[P3]-[SSRS]-[P2])n-[P1])- / CG; CG / -([P2]-[SSRS]-[P3]-[P2])n- / CG; or CG / -([P2]-[P3]-[SSRS]-[P2])n- / CG In some aspects, n is an integer such as 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, n is higher than 10, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0285] In some aspects, the two landing pad plasmids or the two expression plasmids are in a configuration selected from the following: head to head, tail to tail, tail to head and head to tail. In some aspects, each expression plasmid comprises at least a nucleic acid encoding a gene of interest (GOI). In some aspects, all GOIs are the same. In some aspects, all GOIs are different. In some aspects, at least one GOI is different from the rest. In some aspects, the first GOI is the HC of the antibody and the second GOI is the LC of the antibody. In some aspects, at least one expression plasmid is bicistronic or polycistronic. In some aspects, the bicistronic expression plasmid encodes a first GOI comprising the HC of the antibody and a second GOI comprising the LC of the antibody.

[0286] In some aspects, each landing pad plasmid in the dual landing pad is addressable. In some aspects, each addressable landing pad plasmid comprises a pair of SSRS, which can be unique or incompatible. In some aspects, the landing pad plasmid comprises two Lox sites. In some aspects, the Lox sites are Lox P and Lox 511. In some aspects, each landing pad plasmid comprises a Lox site and a Frt site. In some aspects, each landing pad plasmid comprises one or two aat sites, for example, two aatP sites.

[0287] In some aspects, each landing pad plasmid is addressable. In some aspects, each addressable landing pad plasmid comprises a pair of addressable SSRS unique to the landing pad. In some aspects, at least one pair of addressable SSRS is a pair of Lox sites. In some aspects, at least one pair of Lox sites is Lox 511 and Lox P. In some aspects, at least one pair of Lox sites is Loxm3 and Loxm7.

[0288] In some aspects, the methods, cell lines, cells or kits of the present disclosure comprise a first addressable landing pad plasmid comprising a Lox 511 and Lox P pair of Lox sites and a second addressable landing pad plasmid comprising a Lox m3 and Lox m7 pair of Lox sites. In some aspects, each addressable landing pad plasmid comprises an attP site that is not cross-compatible.

[0289] In some aspects, the LoxP site is selected from SEQ ID NOs: 1-11 and 28-82 and any combination thereof. In some aspects, the Frt site is selected from SEQ ID NOs: 12 and 83-91 and any combination thereof. In some aspects, the addressable pad disclosed herein may comprise an SSRS selected from SEQ ID NOs: 1-13 and 28-109 and any combination thereof, or a combination thereof.

[0290] In some aspects, the att site is selected from SEQ ID NOs: 92 to 109 and any combination thereof. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 92 and an attP site of SEQ ID NO: 93. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 94 and an attP site of SEQ ID NO: 95. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 96 and an attP site of SEQ ID NO: 97. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 98 and an attP site of SEQ ID NO: 99. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 100 and an attP site of SEQ ID NO: 101. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 102 and an attP site of SEQ ID NO: 103. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 104 and an attP site of SEQ ID NO: 105. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 106 and an attP site of SEQ ID NO: 107. In some aspects, a pair of att sites includes an attB site of SEQ ID NO: 108 and an attP site of SEQ ID NO: 109. Nuclease

[0291] As used herein, the term "nuclease" refers to an enzyme that has catalytic activity for DNA cleavage.

[0292] In some aspects, a nuclease agent can promote homologous recombination between two plasmids (e.g., linear plasmids) disclosed herein (e.g., a parental plasmid and a landing pad plasmid). In some aspects, the plasmid integrated into the genome of the parental cell line (the parental plasmid, P1) and the landing pad plasmid (P2) contain homology regions, and immediately adjacent to each homology region is a sequence targeted by a nuclease (e.g., a CRISPR / Cas nuclease) that is present in the parental plasmid integrated into the parental cell line but not in the landing pad plasmid to be recombined into the parental cell line.

[0293] The size of the recognition site for a nuclease that mediates homologous recombination can vary and include, for example, a recognition site that is at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 221 0, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 80, at least about 890, at least about 900, at least about 910, at least about 920, at least about 930, at least about 940, at least about 950, at least about 960, at least about 970, at least about 980, at least about 990, at least about 1000, at least about 1010, at least about 1020, at least about 1030, at least about 1040, at least about 1050, at least about 1060, at least about 1070, at least about 1080, at least about 1090, at least about 1100, at least about 1120, at least about 1130, at least about 1140, at least about 1150, at least about 1160, at least about 1170, at least about 1180, at least about 1190, at least about 1200at least about 940, at least about 950, at least about 960, at least about 970, at least about 980, at least about 990, at least about 1000, at least about 1010, at least about 1020, at least about 1030, at least about 1040, at least about 1050, at least about 1060, at least about 1070, at least about 1080, at least about 1090, at least about 1100, at least about 1110, at least about 1120, at least about 1130, at least about 1140, at least about 1150, at least about 1160, at least about 1170, at least about 1180, at least about 1190, at least about 1200, at least about 2010, at least about 2020, or more nucleotides.

[0294] The size of the recognition site for a nuclease that mediates homologous recombination can vary and include, for example, a recognition site that is about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 6, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, about 1000, about 1010, about 1020, about 1030, about 1040, about 1050, about 1060, about 1070, about 1080, about 1090, about 1100, about 1110, about 1120, about 1130, about 1140, about 1150, about 1160, about 1170, about 1180, about 1190, about 1200, about 2010, about 2020 or more nucleotides.

[0295] The size of the recognition site for a nuclease that mediates homologous recombination can vary and include, for example, a recognition site having a length of between about 4 and about 10, between about 10 and about 20, between about 20 and about 30, between about 30 and about 40, between about 40 and about 50, between about 50 and about 60, between about 60 and about 70, between about 70 and about 80, between about 80 and about 90, between about 90 and about 100, between about 100 and about 125, between about 125 and about 150, between about 150 and about 175, between about 175 and about 250. between about 200 and about 225, between about 225 and about 250, between about 250 and about 275, between about 275 and about 300, between about 300 and about 325, between about 325 and about 350, between about 350 and about 375, between about 375 and about 400, between about 400 and about 425, between about 425 and about 450, between about 450 and about 475, between about 475 and about 500, between about 500 and about 525, between about 525 and about 550, between about 550 and about 575, between about 575 and about 600, between about 600 and about 625, between about 625 and about 650, between about 650 and about 675, between about 675 and about 700, between about 700 and about 725, between about 725 and about 750, between about 750 and about 775, between about 775 and about 800, between about 800 and about 825, between about 825 and about 850, between about 850 and about 875, between about 875 and about 900, between about 900 and about 925, and between about 925 and about 950. , between about 950 and about 975, between about 975 and about 1000, between about 1000 and about 1100, between about 1100 and about 1200, between about 1200 and about 1300, between about 1300 and about 1400, between about 1400 and about 1500, between about 1500 and about 1600, between about 1600 and about 1700, between about 1700 and about 1800, between about 1800 and about 1900, between about 1900 and about 2000, or between about 2000 and about 2100, or more nucleotides.

[0296] On the one hand, each monomer of nuclease recognizes the recognition site of at least 9 nucleotides.In other aspects, the length of recognition site is about 9 to about 12 nucleotides, about 12 to about 15 nucleotides, about 15 to about 18 nucleotides or about 18 to about 21 nucleotides, and any combination of such sub-ranges (for example, 9-18 nucleotides).Recognition site can be palindromic, i.e., the sequence on a chain reads the same in opposite directions on the complementary chain.It should be recognized that a given nuclease can bind to the recognition site and cut the binding site, or alternatively, the nuclease can be combined with a sequence different from the recognition site.In addition, no matter whether the nick / cut site is within or outside the nuclease binding site, the term recognition site includes both the nuclease binding site and the nick / cut site.In another variation, the cutting by the nuclease can occur at the nucleotide position directly opposite to each other to produce a flat end cutting, or in other cases, the nick can be staggered to produce a single-stranded overhang (also referred to as "sticky end"), which can be a 5' overhang or a 3' overhang.

[0297] In some aspects, one of the sequences in the parental plasmid that is lost in the landing pad plasmid is SEQ ID NO:14, and the other is SEQ ID NO:15.

[0298] In some aspects, one of the sequences in the parental plasmid that is lost in the landing pad plasmid is SEQ ID NO:14.

[0299] In some aspects, one of the sequences in the parental plasmid that is lost in the landing pad plasmid is SEQ ID NO:15.

[0300] Any nuclease that induces a nick or double-strand break in the desired recognition site can be used in the methods and compositions disclosed herein. Naturally occurring or natural nucleases can be used, as long as the nuclease induces a nick or double-strand break in the desired recognition site. Alternatively, modified or engineered nucleases can be used. "Engineering nucleases" include nucleases that are engineered (modified or derived) from their native form to specifically recognize and induce a nick or double-strand break in the desired recognition site. Therefore, engineered nucleases can be derived from natural, naturally occurring nucleases, or it can be artificially produced or synthesized. The modification of nucleases can be as little as one amino acid in a protein cutting agent or one nucleotide in a nucleic acid cutting agent. In some aspects, engineered nucleases induce a nick or double-strand break in a recognition site, wherein the recognition site is not a sequence recognized by a natural (unengineered or unmodified) nuclease. Producing a nick or double-strand break in a recognition site or other DNA can be referred to as a "cutting" or "cleaving" recognition site or other DNA in this article. Homologous recombination system

[0301] In some aspects of the disclosure, homologous recombination is mediated by a CRISPR / Cas system, a TALEN system, a ZFN system, a meganuclease, or a restriction endonuclease. CRISPR / Cas

[0302] In some aspects, the nuclease for homologous recombination in the various methods and compositions disclosed herein may include a CRISPR / Cas system. Note that the depiction of CRISPR / Cas as a "default" homologous recombination system in the figure is merely exemplary, and the process illustrated in the figure can be performed using an alternative homologous recombination system (e.g., a TALEN system, a ZFN system, a mega-nuclease, or a restriction endonuclease). Such CRISPR / Cas systems can use, for example, Cas9 nucleases, which in some cases are codon-optimized for the desired cell type to be expressed therein. Such systems can also use a guide RNA (gRNA) comprising two separate molecules. Exemplary two-molecule gRNAs include crRNA-like ("CRISPR RNA" or "target RNA" or "crRNA" or "crRNA repeats") molecules and corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator RNA" or "tracrRNA" or "scaffold") molecules.

[0303] The crRNA comprises a DNA targeting segment (single strand) of the gRNA and a section of nucleotides of half of the double-stranded RNA (dsRNA) duplex forming the protein binding segment of the gRNA. The corresponding tracrRNA (activator RNA) comprises a section of nucleotides of the other half of the dsRNA duplex forming the protein binding segment of the gRNA. Therefore, the nucleotide segment of the crRNA is complementary to the nucleotide segment of the tracrRNA and hybridizes to form the dsRNA duplex of the protein binding domain of the gRNA. Therefore, it can be said that each crRNA has a corresponding tracrRNA. The crRNA additionally provides a single-stranded DNA targeting segment. Therefore, the gRNA comprises a sequence and tracrRNA hybridized with the target sequence. Therefore, crRNA and tracrRNA (as a corresponding pair) hybridize to form gRNA. If used for intracellular modification, the exact sequence and / or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecule will be used.

[0304] The naturally occurring genes encoding the three elements (Cas9, tracrRNA and crRNA) are usually organized in one or more operons. Naturally occurring CRISPR RNA is different according to the Cas9 system and the organism, but generally contains a targeting segment between 21 and 72 nucleotides in length, and its flanking length is two directional repeats (DR) between 21 and 46 nucleotides (see, for example, WO 2014 / 131833). In the case of Streptococcus pyogenes (S.pyogenes), DR is 36 nucleotides long and the targeting segment is 30 nucleotides long. The DR at 3' is complementary to the corresponding tracrRNA and hybridizes, and the latter then binds to the Cas9 protein.

[0305] Alternatively, the system further uses the crRNA-tracrRNA construct (i.e., a single transcript) of the fusion that functions together with the codon-optimized Cas9. The single RNA is generally referred to as guide RNA or gRNA. In gRNA, the crRNA portion is identified as the "target sequence" of a given recognition site, and tracrRNA is generally referred to as a "scaffold". In short, a short DNA fragment containing a target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid includes a target sequence (in some aspects, about 20 nucleotides), a form of tracrRNA sequence (scaffold), and a suitable promoter active in the cell and the necessary elements for correct processing in eukaryotic cells. Many systems rely on customized complementary oligonucleotides, which are annealed to form double-stranded DNA and then cloned into the gRNA expression plasmid.

[0306] Then the gRNA expression cassette and the Cas9 expression cassette are introduced into the cell. See, e.g., Mali P et al. (2013) Science 2013 Feb 15; 339(6121):823-6; Jinek M et al. Science 2012 Aug 17; 337(6096):816-21; Hwang WY et al. Nat Biotechnol 2013 Mar; 31(3):227-9; Jiang W et al. Nat Biotechnol 2013 Mar; 31(3):233-9; and Cong L et al. Science 2013 Feb 15; 339(6121):819-23, each of which is incorporated herein by reference. See also, e.g., WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, and WO / 2013142578A1, each of which is incorporated herein by reference.

[0307] In some aspects, the Cas9 nuclease can be provided in the form of a protein. In some aspects, the Cas9 protein can be provided in the form of a complex with a gRNA. In other aspects, the Cas9 nuclease can be provided in the form of a nucleic acid encoding a protein. The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA. In some aspects, the gRNA can be provided in the form of RNA. In other aspects, the gRNA can be provided in the form of a DNA encoding RNA. In some aspects, the gRNA can be provided in the form of a separate crRNA and tracrRNA molecule or a separate DNA molecule encoding crRNA and tracrRNA, respectively.

[0308] In one aspect, the method for generating a landing pad cell disclosed herein further comprises introducing into the cell: (a) a first expression construct comprising a first promoter operably linked to a first nucleic acid sequence encoding a CRISPR-associated (Cas) protein; (b) a second expression construct comprising a second promoter operably linked to a genomic target sequence linked to a guide RNA (gRNA), wherein the genomic target sequence is flanked by a protospacer adjacent motif. Optionally, the genomic target sequence is flanked by a protospacer adjacent motif (PAM) sequence at the 3' end.

[0309] In some aspects, the gRNA comprises a third nucleic acid sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In one aspect, the Cas protein is a type I Cas protein. In one aspect, the Cas protein is a type II Cas protein. In one aspect, the type II Cas protein is Cas9. In one aspect, type II Cas (e.g., Cas9) is a human codon-optimized Cas.

[0310] In some aspects, Cas proteins are "nickases" that can produce single-strand breaks (i.e., "nicks") at the target site without cutting the two strands of double-stranded DNA (dsDNA). For example, Cas9 contains two nuclease domains: a RuvC-like nuclease domain and an HNH-like nuclease domain, which are responsible for cutting the relative DNA strands. Mutations in any of these domains can produce nickases. Examples of mutations that produce nickases can be found in, for example, WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference.

[0311] In some aspects, two separate Cas proteins (e.g., nickases) with specificity for target sites on each strand of dsDNA can produce protruding sequences complementary to protruding sequences on another nucleic acid or separate regions on the same nucleic acid. The protruding ends produced by contacting the nucleic acid with two nickases with specificity for target sites on two strands of dsDNA can be 5' or 3' protruding ends. For example, a first nickase can produce single-strand breaks on the first strand of dsDNA, and a second nickase can produce single-strand breaks on the second strand of dsDNA, thereby producing protruding sequences. The target site of each nickase that produces single-strand breaks can be selected so that the protruding end sequence produced is complementary to the protruding end sequence on different nucleic acid molecules. The complementary protruding ends of two different nucleic acid molecules can be annealed by the method disclosed herein. In some aspects, the target site of the nickase on the first strand is different from the target site of the nickase on the second strand.

[0312] In some aspects, the first nucleic acid comprises a mutation that destroys at least one amino acid residue in the nuclease active site in the Cas protein, wherein the mutant Cas protein produces a break only in one chain of the target DNA region, and wherein the mutation reduces the non-homologous recombination in the target DNA region. On the one hand, the first nucleic acid encoding the Cas protein also comprises a nuclear localization signal (NLS). On the one hand, the nuclear localization signal is an SV40 nuclear localization signal. TALEN

[0313] In some aspects, the nuclease agent for homologous recombination in the various methods and compositions disclosed herein may include a TALEN system. Therefore, on the one hand, the nuclease agent is a transcription activator-like effector nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to produce double-strand breaks at specific target sequences in the genome of prokaryotes or eukaryotes. TAL effector nucleases are produced by fusing a natural or engineered transcription activator-like (TAL) effector or a functional portion thereof with a catalytic domain of an endonuclease (e.g., such as FokI).

[0314] The unique modular TAL effector DNA binding domain allows the design of proteins with potential recognition specificity for any given DNA. Therefore, the DNA binding domain of the TAL effector nuclease can be engineered to recognize specific DNA target sites and thus used to generate double-strand breaks at the desired target sequence. See, WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze and Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148; all of which are incorporated herein by reference.

[0315] Examples of suitable TAL nucleases and methods for making suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Nos. 2011 / 0239315A1, 2011 / 0269234A1, 2011 / 0145940A1, 2003 / 0232410A1, 2005 / 0208489A1, 2005 / 0026157A1, 2005 / 0064474A1, 2006 / 0188987A1, and 2006 / 0063231A1 (each of which is hereby incorporated by reference).

[0316] In various aspects, TAL effector nucleases are engineered to cut in or near a target nucleic acid sequence, e.g., in a genomic locus of interest, where the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. TAL nucleases suitable for use with the various methods and compositions provided herein include those specifically designed to bind at or near a target nucleic acid sequence to be modified by a targeting vector as described herein.

[0317] On the one hand, each monomer of TALEN comprises 12-25 TAL repeat sequences, wherein each TAL repeat sequence binds to a 1bp subsite. On the one hand, the nuclease agent is a chimeric protein comprising a TAL repeat sequence-based DNA binding domain operably linked to an independent nuclease. On the one hand, the independent nuclease is a FokI nuclease. On the one hand, the nuclease agent comprises a first TAL repeat sequence-based DNA binding domain and a second TAL repeat sequence-based DNA binding domain, wherein the first and second TAL repeat sequence-based DNA binding domains are each operably linked to the FokI nuclease, wherein the first and second TAL repeat sequence-based DNA binding domains recognize two continuous target DNA sequences separated by about 6bp to about 40bp cleavage sites in each chain of the target DNA sequence, and wherein the FokI nuclease dimerizes and produces a double-stranded break at the target sequence.

[0318] In one aspect, the nuclease agent comprises a first TAL repeat-based DNA binding domain and a second TAL repeat-based DNA binding domain, wherein the first and second TAL repeat-based DNA binding domains are each operably linked to a FokI nuclease, wherein the first and second TAL repeat-based DNA binding domains recognize two consecutive target DNA sequences in each strand of the target DNA sequence separated by approximately a 5 bp or 6 bp cleavage site, and wherein the FokI nuclease dimerizes and produces a double-stranded break. Zinc Finger Nuclease (ZFN)

[0319] In some aspects, the nuclease agent for homologous recombination in the various methods and compositions disclosed herein may include a zinc finger nuclease (ZFN) system. On the one hand, each monomer of ZFN includes 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain is combined with a 3bp subsite. In other aspects, ZFN is a chimeric protein comprising a zinc finger-based DNA binding domain operably connected to an independent nuclease. On the one hand, the independent endonuclease is a FokI endonuclease. On the one hand, the nuclease agent includes the first ZFN and the second ZFN, wherein each of the first ZFN and the second ZFN is operably connected to the FokI nuclease, wherein the first and second ZFNs recognize two continuous target DNA sequences separated by about 6bp to about 40bp cleavage sites or about 5bp to about 6bp cleavage sites in each chain of the target DNA sequence, and wherein the FokI nuclease dimerizes and produces double-strand breaks. See, e.g., US20060246567; US20080182332; US20020081614; US20030021776; WO / 2002 / 057308A2; US20130123484; US20100291048; and WO / 2011 / 017293A2, each of which is incorporated herein by reference. Meganuclease

[0320] In some aspects, the nuclease agents used for homologous recombination in the various methods and compositions disclosed herein can include a mega-nuclease system. Mega-nucleases have been divided into four families based on conserved sequence motifs, which are the "LAGLIDADG", "GIY-YIG", "HNH" and "His-Cys box" families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds.

[0321] HEase is known for its long recognition site and tolerance to some sequence polymorphisms in its DNA substrate. Meganuclease domains, structures and functions are known, see, e.g., Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764.

[0322] In some examples, naturally occurring variants and / or engineered derivatives of meganucleases are used. Methods for modifying kinetics, cofactor interactions, expression, optimal conditions and / or recognition site specificity and screening for activity are known, see, e.g., Epinat et al., (2003) Nucleic Acids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:el78; Smith et al., (2006) Nucleic Acids Res 34:el49; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:el54; WO 2005105989; WO2003078619; WO 2006097854; WO 2006097853; WO 2006097784; and WO 2004031346.

[0323] Any meganuclease that can be used in this article includes, but is not limited to, I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SecVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP, PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII or any active variant or fragment thereof.

[0324] In one aspect, the meganuclease recognizes a double-stranded DNA sequence of 12 to 40 base pairs. In one aspect, the meganuclease recognizes a perfectly matched target sequence in one of the heterologous plasmids described herein. In one aspect, the meganuclease is a homing nuclease. In one aspect, the homing nuclease is a "LAGLIDADG" family of homing nucleases. In one aspect, the "LAGLIDADG" family of homing nucleases is selected from I-Scel, I-Crel, and I-Dmol. Restriction endonucleases

[0325] In some respects, the nuclease agent for homologous recombination in various methods and compositions disclosed herein can include restriction endonucleases, which include type I, type II, type III and type IV nucleases. Type I and type III restriction endonucleases recognize specific recognition sites, but usually cut at variable positions away from the nuclease binding site, and the position can be hundreds of base pairs away from the cutting site (recognition site). In type II systems, restriction enzyme activity is independent of any methylase activity, and cutting usually occurs in a specific site within or near the binding site. Most type II enzymes cut palindromic sequences, but type IIa enzymes recognize non-palindromic recognition sites and cut outside the recognition site, type IIb enzymes cut sequences twice, wherein both sites are outside the recognition site, and type IIs enzymes recognize asymmetric recognition sites, and cut at a limited distance of about 1-20 nucleotides from the recognition site on one side. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example, in the REBASE database (webpage at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) Mobile DNA II, pp. 761-783, Craigie et al., eds., (ASM Press, Washington, DC).

[0326] Nucleases can be introduced into cells by any method known in the art. Polypeptides encoding nucleases can be directly introduced into cells. Alternatively, polynucleotides encoding nucleases can be introduced into cells. When polynucleotides encoding nucleases are introduced into cells, nucleases can be expressed transiently, conditionally or constitutively in cells. Therefore, polynucleotides encoding nucleases can be included in expression cassettes and operably connected to conditional promoters, inducible promoters, constitutive promoters or tissue-specific promoters. Such target promoters are discussed in further detail elsewhere herein. Alternatively, nucleases are introduced into cells as mRNA encoding or comprising nucleases.

[0327] Also provided are active variants and fragments of nucleases (i.e., engineered nucleases). Such active variants may include at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with natural nucleases, wherein the active variant retains the ability to cut at the desired recognition site, and thus retains the nick or double-strand break inducing activity. For example, any nuclease described herein may be modified from a natural endonuclease sequence, and is designed to recognize and induce a nick or double-strand break at a recognition site not recognized by a natural nuclease. Therefore, in some aspects, engineered nucleases have the specificity of inducing a nick or double-strand break at a recognition site different from the corresponding natural nuclease recognition site. The determination of nick or double-strand break inducing activity is known, and the overall activity and specificity of endonucleases to DNA substrates containing recognition sites are usually measured.

[0328] When providing a nuclease agent to a cell by introducing a polynucleotide encoding a nuclease agent, the polynucleotide encoding the nuclease agent can be modified to replace codons with a higher frequency of use in the target cell compared to the naturally occurring polynucleotide sequence encoding the nuclease agent. For example, the polynucleotide encoding the nuclease agent can be modified to replace codons with a higher frequency of use in a given target prokaryotic or eukaryotic cell (including bacterial cells, yeast cells, human cells, non-human cells, non-rat eukaryotic cells, mammalian cells, rodent cells, non-rat rodent cells, mouse cells, rat cells, hamster cells or any other target host cell) compared to the naturally occurring polynucleotide sequence. Homologous recombination sequence

[0329] A key advantage of the methods and compositions of the present disclosure is the possibility to generate landing pad cells without the need for information about the genomic context in which the landing pad plasmid or a portion thereof is to be inserted. This is possible because the methods disclosed herein rely on the targeted incorporation of a landing pad plasmid or a portion thereof in a position occupied by a parent plasmid. Sequence information about parent plasmids (e.g., commercial plasmids) is generally available or known in the art. Therefore, it is possible to rely on such information to generate homologous recombination sequences that will direct the exchange of internal subsequences of the parent plasmid with landing pad plasmid sequences or a portion thereof via homologous recombination.

[0330] In some aspects of the disclosure, the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO:16.

[0331] In some aspects of the disclosure, the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO:17.

[0332] In some aspects of the disclosure, the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO:16, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO:17.

[0333] In some aspects, the 5' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, or at least about 310 from SEQ ID NO: 16. 530, at least about 540, at least about 550, at least about 553, or at least about 560, at least about 570, at least about 580, at least about 590, at least about 600, at least about 610, at least about 620, at least about 630, at least about 640, at least about 650, at least about 660, at least about 670, at least about 680, at least about 690, at least about 700, at least about 710, at least about 720, at least about 730, at least about 740, at least about 750, at least about 760, at least about 770,

[0334] In some aspects, the 3' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, or more of the 3' homologous recombination sites from SEQ ID NO: 17. , at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 560, at least about 570, at least about 580, at least about 590, at least about 500 about 600, at least about 610, at least about 620, at least about 630, at least about 640, at least about 650, at least about 660, at least about 670, at least about 680, at least about 690, at least about 700, at least about 710, at least about 720, at least about 730, at least about 740, at least about 750, at least about 760, at least about 770, at least about 780, at least about 790, at least about 800, at least about 810, at least about 820, at least about 830, at least about 840, at least about 850, at least about 860, at least about 870, at least about 880, at least about 890 100, at least about 1010, at least about 1020, at least about 1030, at least about 1040, at least about 1050, at least about 1060, at least about 1070, at least about 1080, at least about 1090, at least about 1100, at least about 1110, at least about 1120, at least about 1130, at least about 1140, at least about 1150, at least about 1160, at least about 1170, at least about 1180, at least about 1190, at least about 1200, at least about 1210, at least about 1220, or at least about 1221 consecutive nucleotides.

[0335] In some aspects, the 5' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, or at least about 310 from SEQ ID NO: 16. 470, at least about 480, at least about 490, at least about 500, at least about 510, at least about 520, at least about 530, at least about 540, at least about 550, at least about 553, or at least about 560, contiguous nucleotides;and the 3' homologous recombination site comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about...

Claims

1. A method for selecting a parent cell suitable for developing a landing pad cell line, the method comprising: (i) Screening and selecting cell lines with high expression titers of target genes (GOI); as well as (ii) further screening the cells of (i) and selecting cells having a low copy number of the parental plasmid comprising a nucleic acid encoding the GOI, wherein the copy number is one or two.

2. The method of claim 1, wherein the parent plasmid comprises two site-specific recombination sites (SSRS), one SSRS, or no SSRS.

3. A method for selecting landing pad cells, the method comprising: (i) screening a parental cell line for the loss of the parental plasmid or a portion thereof, and selecting cells having such a loss (deletion); as well as (ii) further screening the cells of (i) for the presence of a landing pad, and selecting cells in which the landing pad is present.

4. A method for selecting landing pad cells, the method comprising: (i) screening a parental cell line for the loss of at least one parental plasmid or a portion thereof, and selecting cells having such a loss (deletion); as well as (ii) further screening the cells of (i) for the presence of at least one landing pad, and selecting cells in which the landing pad is present.

5. The method of claim 3 or claim 4, further comprising screening the landing pad sequence in the landing pad cell for a feature selected from the group consisting of: (i) the presence or absence of low-complexity regions or high-complexity regions; (ii) the presence or absence of retrotransposon sequences; (iii) the presence or absence of Alu repeat sequences; (iv) the presence or absence of long interspersed nuclear elements (LINEs); (v) presence or absence of CpG islands; (vi) the level of cytosine methylation; (vii) the level of histone acetylation; (viii) the presence or absence of active transcription; as well as (ix) any combination thereof.

6. A method for generating landing pad cells, the method comprising (i) deleting at least one parent plasmid comprising the first GOI or a part thereof in the parent cell line, and (ii) introducing a landing pad plasmid or a portion thereof comprising a landing pad into said cell following said at least one deletion.

7. The method of claim 6, wherein the landing pad plasmid or a portion thereof comprising a landing pad is inserted at the site of the deletion of (i).

8. The method of claim 6, wherein the landing pad plasmid or a portion thereof comprising a landing pad is inserted at a site that is not a deletion of (i).

9. A method for generating landing pad cells, the method comprising: The landing pad plasmid is integrated into the genome of the parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is located in the parent plasmid Each landing pad plasmid contains (1) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1); and (3) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2), which are homologous to the corresponding homologous recombination sites in the parent plasmid; and The homologous recombination sites of the landing pad plasmid recombined with the corresponding homologous recombination sites of the parent plasmid, thereby integrating the landing pad plasmid at an internal position within the parent plasmid inserted into the genomic DNA of the parent cell.

10. The method according to any one of claims 6 to 9, wherein the parent plasmid is located in more than one genomic locus.

11. A method for identifying landing pad cells, the method comprising (1) removing at least a portion of the first GOI from a parent plasmid integrated into the genomic sequence of a parent cell; (2) integrating the landing pad plasmid at an alternative genomic locus; (3) screening a library of candidate cells comprising at least one copy of the landing pad plasmid integrated at at least one alternative genomic locus, wherein the candidate cell lines are evaluated for one or more of the following properties: (a) the cell titer exceeds a predetermined threshold level; (b) the landing pad plasmid or the landing pad copy number is at a predetermined value; (c) RNA expression level exceeds a predetermined threshold level; (d) multiple plasmid copies, if present, having a specific plasmid configuration; (e) at least a portion of the first GOI of the parent plasmid is deleted; and (f) There is at least one landing pad with a functional SSRS.

12. The method of claim 11, wherein the parental cell is a historical cell line.

13. The method of claim 11, wherein the library of candidate cells is a library generated via random integration of the landing pad sequence at multiple locations in the genome of a parent cell.

14. The method according to any one of claims 11 to 13, wherein the method selects thermal cells having the landing pad sequence integrated in a hotspot.

15. The method according to any one of claims 11 to 14, wherein the parent cell line is a CHO cell line.

16. A method of generating an expression cell, the method comprising integrating a second GOI plasmid into the genome of a landing pad cell according to claims 3-15 using a site-specific recombinase recombination, wherein the resulting expression plasmid comprises (1) a polynucleotide sequence comprising a nucleic acid encoding a second GOI; and (2) two SSRS flanking the polynucleotide of (1); The site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the second GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

17. A method for generating an expressing cell, the method comprising: (a) integrating the landing pad plasmid or a portion thereof into the genome of a parent cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is located in the parent plasmid, wherein each landing pad plasmid or portion thereof comprises (1a) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2a) two SSRS flanking the polynucleotide sequence of (1a); and (3a) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2a), which are homologous to the corresponding homologous recombination sites in the parent plasmid; and wherein homologous recombination sites of the landing pad plasmid or a portion thereof recombine with corresponding homologous recombination sites of the parent plasmid in a landing pad at a different genomic locus, thereby integrating the landing pad plasmid or a portion thereof at an internal position within the landing pad at the different genomic locus in the genomic DNA of the parent cell; and (b) integrating a second GOI plasmid into the genome of the landing pad cells using a site-specific recombinase, wherein the expression plasmid comprises (1b) a polynucleotide sequence comprising a nucleic acid encoding a GOI; and (2b) two SSRS flanking the polynucleotide of (1b); The site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

18. A method for generating landing pad cells, the method comprising: (a) removing at least a portion of a parent plasmid from a first hotspot location in a parent cell line; as well as (b) integrating a landing pad plasmid into a second hotspot location in the genome of the parent cell at a targeted integration site using homologous recombination or random integration, wherein the sequence targeted for homologous recombination or random integration is present in the landing pad plasmid Each landing pad plasmid contains (1) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1); and (3) Two homologous recombination sites located at the 5' and 3' ends of the SSRS of (2), which are homologous to the corresponding homologous recombination sites in the genome of the parental cell line.

19. A method for generating an expressing cell, the method comprising: (a) removing a parent plasmid or a portion thereof from a first hotspot location in a parent cell line, (b) integrating the landing pad plasmid into a second hotspot location in the genome of the parental cell at a targeted integration site using homologous recombination, wherein the sequence targeted for homologous recombination is present in the parental cell line Each landing pad plasmid contains (1b) a polynucleotide sequence comprising a nucleic acid encoding at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; (2b) two site-specific recombination sites (SSRS) flanking the polynucleotide sequence of (1b); and (3b) two homologous recombination sites located at the 5' and 3' ends of the SSRS with respect to (2b), which are homologous to the corresponding homologous recombination sites in the parental cell line, wherein the homologous recombination sites of the landing pad plasmid recombined with corresponding homologous recombination sites of the parental cell line, thereby integrating the landing pad plasmid at an internal location within the genomic DNA of the parental cell, and (c) integrating the GOI plasmid into the genome of the landing pad cells using a site-specific recombinase, wherein the expression plasmid comprises (1c) a polynucleotide sequence comprising a nucleic acid encoding a first GOI; and (2c) two SSRS flanking the polynucleotide of (1c); The site-specific recombination site of the landing pad plasmid recombines with the corresponding site-specific recombination site of the GOI plasmid, thereby integrating the GOI plasmid at an internal position within the landing pad plasmid of the landing pad cell.

20. The method of any one of claims 1-19, wherein the landing pad cells comprise a plasmid having a topology corresponding to: CG1 / -[P1]-[P2]-[SSRS]-[M]-[SSRS]-[P2]-[P1]- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-[P2]-[P1]- / CG2; CG1 / -[P1]- / CG2; CG1 / -([P1]-([P2])n-[P1])- / CG2; -[P1]-[P2]-[P1]-; or -([P1]-([P2])n-[P1])- in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; and [SSRS] is site-specific recombination site (SSRS); n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. The method according to any one of claims 16, 17 or 19, wherein the topology of the plasmid integrated in the expression cell corresponds to the following description: CG1 / -[P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[P3]-[=SSRS]-[P2])n-[P1]- / CG2 CG1 / -([P2]-[P3]-[SSRS]-[P2])n- / CG2; in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid containing a gene of interest (GOI); and [SSRS] is site-specific recombination site (SSRS); n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

22. The method according to any one of claims 9 or 17-21, wherein the homologous recombination is mediated by a CRISPR / Cas system, a TALEN system or a ZFN system.

23. The method of claim 22, wherein the CRISPR / Cas system further comprises a single guide RNA (sgRNA).

24. The method of any one of claims 2 or 9-23, wherein the site-specific recombinase recombination site (SSRS) is a Tyr recombinase site, a Tyr integrase site, a serine resolvase / invertase site, or a serine integrase site.

25. The method of claim 24, wherein the Tyr recombinase site comprises a Cre, Dre, Flp, KD, B3 or B3 Tyr recombinase site.

26. The method of claim 24, wherein the Tyr integrase site comprises a λ (lambda), HK022 or HP1 Tyr integrase site.

27. The method of claim 24, wherein the serine resolvase / invertase site comprises a γδ (gamma delta), ParA, Tn3, or Gin serine resolvase / integrase site.

28. The method of claim 24, wherein the serine integrase site comprises PhiC31, Bxb1, pr R4 serine integrase site.

29. The method of claim 24, wherein the Tyr recombinase site comprises a Cre Tyr recombinase site.

30. The method of claim 24, wherein the SSRS is a LoxP site.

31. The method of claim 30, wherein the LoxP site comprises the nucleic acid sequence shown in SEQ ID NO: 1 (wild-type LoxP).

32. The method of claim 30, wherein the LoxP site comprises a mutant LoxP site.

33. The method of claim 32, wherein the mutant LoxP site comprises the nucleic acid sequence shown in SEQ ID NO: 2 (Mutant LoxP).

34. The method of claim 32, wherein the mutant LoxP site comprises a nucleic acid selected from the group consisting of: SEQ ID NO: 3 (Lox 511); SEQ ID NO: 4 (Lox 5171); SEQ ID NO: 5 (Lox 2272); SEQ ID NO: 6 (Lox M2); SEQ ID NO: 7 (Lox M3); SEQ ID NO: 8 (Lox M7); SEQ ID NO: 9 (Lox M11); SEQ ID NO: 10 (Lox 71); and SEQ ID NO: 11 (Lox 66).

35. The method of claim 24, wherein the Tyr recombinase site comprises a Flp Tyr recombinase site.

36. The method of claim 35, wherein the SSRS is a short flippase recognition target (FRT) site.

37. The method of claim 24, wherein the serine integrase site comprises an attP or attB site.

38. The method according to any one of claims 9, 10 or 17-37, wherein the at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker is glutamine synthetase (GS) and / or dihydrofolate reductase (DHFR).

39. The method according to any one of claims 9, 10 or 17-38, wherein the at least one selection marker and / or at least one nucleic acid sequence encoding a detectable marker is a drug resistance gene.

40. The method of claim 39, wherein the drug resistance gene is an antibiotic resistance gene.

41. The method of claim 40, wherein the antibiotic resistance gene is a puromycin resistance gene.

42. The method of claim 41, wherein the puromycin resistance gene is puromycin-N-acetyltransferase.

43. The method of any one of claims 9, 10, or 17-42, wherein the at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker comprises a protein.

44. The method of claim 43, wherein the protein is a fluorescent protein.

45. The method of claim 44, wherein the fluorescent protein is mCherry.

46. ​​The method of claim 44, wherein the fluorescent protein comprises GFP, ZsGreen1, AcGFP1, EGFP, GFPuv, AcGFP, EBFP, EYFP, ECFP, tdTomato, mCherry, DsRed, AmCyan, ZsGreen, ZsYellow, DsRed2, DsRed-Express, HcRed, AsRed, mOrange, mOrange2, mPlum, mStrawberry, mBanana, YFP, mRaspberry, HcRed1, E2-Crimson, or any combination thereof.

47. The method of any one of claims 1 to 46, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

48. The method of any one of claims 1 to 46, wherein the cell is HEK293 or NS0.

49. The method of any one of claims 1, 2, 6-8, 11-17 or 19-48, wherein the nucleic acid encoding the GOI encodes at least one polypeptide.

50. The method of claim 49, wherein the at least one polypeptide is an antibody or a fusion protein.

51. The method of any one of claims 16, 17, or 19-50, wherein the expression plasmid comprises one, two, or more than two copies of the GOI, detectable marker, or a combination thereof.

52. The method of claim 51, further comprising determining the expression of the GOI, detectable marker, or a combination thereof.

53. The method of claim 52, wherein the expression of the GOI is determined quantitatively and / or qualitatively.

54. The method of claim 52 or claim 53, wherein expression of the GOI is determined by cell sorting, FACS, cell surface staining, Western blot, Northern blot, column chromatography, capillary electrophoresis, microfluidics, UV absorbance, immunohistochemistry, cell size, secreted protein levels, transcript levels, or any combination thereof.

55. The method of any one of claims 3 to 54, wherein the landing pad plasmid or expression plasmid is integrated into the genome of the cell at 1 copy number.

56. The method of any one of claims 3 to 55, wherein the landing pad plasmid or expression plasmid is integrated into the genome of the cell at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

57. The method of any one of claims 9-10 or 17-56, wherein (i) the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 18 or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 19 or a subsequence thereof; (ii) the 5' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 114 or a subsequence thereof, and the 3' homologous recombination site comprises the polynucleotide sequence of SEQ ID NO: 115 or a subsequence thereof; or (iii) a 5' homologous recombination site and a 3' homologous recombination site comprise polynucleotide sequences flanking the parent plasmid.

58. The method of any one of claims 1 to 57, wherein the parent plasmid comprises an open reading frame (ORF) encoding a first GOI, such as an antibody.

59. A landing pad cell comprising a plasmid having a topology corresponding to the following description: CG1 / -[P1]-[P2]-[SSRS]-[M]-[SSRS]-[P2]-[P1]- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[M]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[M]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -([P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1])- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[M]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[M]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-[P2]-[P1]- / CG2; CG1 / -[P1]- / CG2; CG1 / -([P1]-([P2])n-[P1])- / CG2; -[P1]-[P2]-[P1]-; or -([P1]-([P2])n-[P1])- in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [M] is a polynucleotide sequence comprising at least one selectable marker and / or at least one nucleic acid sequence encoding a detectable marker; and [SSRS] is site-specific recombination site (SSRS); n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

60. An expression cell comprising a plasmid having a topology corresponding to the following description: CG1 / -[P1]-([P2]-[SSRS]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[SSRS]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[SSRS]-[P3]-[P2])n-[P1]- / CG2; CG1 / -([P2]-[SSRS]-[P3]-[P2])n- / CG2; CG1 / -[P1]-([P2]-[P3]-[SSRS]-[P2])n-[P1]- / CG2; or CG1 / -([P2]-[P3]-[SSRS]-[P2])n- / CG2; in CG1 and CG2 are parental cell genomic sequences that flank the inserted plasmid; [P1] is a polynucleotide sequence derived from the parental plasmid; [P2] is a polynucleotide sequence derived from the landing pad plasmid; [P3] is a polynucleotide sequence derived from a plasmid containing a gene of interest (GOI); and [SSRS] is site-specific recombination site (SSRS); n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

61. A cell line produced by the method of any one of claims 3 to 60.

62. A kit comprising the cell of claim 61 or a cell produced according to the method of any one of claims 1 to 61 and instructions for their use.

63. An isolated cell comprising a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI) integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or polynucleotide subsequence within SEQ ID NO:20 or SEQ ID NO:

116.

64. A method comprising introducing a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI) into a CHO cell and obtaining the CHO cell, wherein the nucleic acid is integrated at a specific locus in the genome of the CHO cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO:

116.

65. A method comprising providing a cell comprising a polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), wherein the polynucleotide sequence comprises the nucleic acid encoding the gene of interest (GOI) operably linked to a promoter, wherein the nucleic acid is integrated at a specific locus in the genome of the CHO cell, and wherein the locus is a nucleotide position or polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO:

116.

66. An isolated cell according to claim 63 or a method according to claim 64 or 65, wherein (i) the nucleotide subsequence within SEQ ID NO:20 comprises the sequence shown in SEQ ID NO:21; or (ii) the nucleotide subsequence within SEQ ID NO:116 comprises the sequence shown in SEQ ID NO:

117.

67. The isolated cell of claim 63 or the method of claim 64 or 65, wherein (i) the nucleotide subsequence from SEQ ID NO:20 consists of the sequence shown in SEQ ID NO:21; or (ii) the nucleotide subsequence from SEQ ID NO:116 consists of the sequence shown in SEQ ID NO:

117.

68. The isolated cell of claim 63 or the method of claim 64 or 65, wherein (i) the nucleotide subsequence from SEQ ID NO:20 is a subsequence upstream (towards the 5' end of SEQ ID NO:20) of the sequence shown in SEQ ID NO:21; or (ii) the nucleotide subsequence from SEQ ID NO:116 is a subsequence upstream (towards the 5' end of SEQ ID NO:116) of the sequence shown in SEQ ID NO:

117.

69. The isolated cell of claim 63 or the method of claim 64 or 65, wherein (i) the nucleotide subsequence from SEQ ID NO:20 is a subsequence downstream (towards the 3' end of SEQ ID NO:20) of the sequence shown in SEQ ID NO:21, or (ii) the nucleotide subsequence from SEQ ID NO:116 is a subsequence downstream (towards the 3' end of SEQ ID NO:116) of the sequence shown in SEQ ID NO:

117.

70. The method of any one of claims 1-58 or 64-69, the cell of claims 59-60, 63 or 66-69, the cell line of claim 61 or the kit of claim 62, wherein the method, the cell, the cell line or the kit comprises at least two landing pad plasmids or at least two expression plasmids.

71. The method, cell, cell line or kit of claim 70, wherein the two landing pad plasmids or the two expression plasmids are in a configuration selected from the group consisting of head to head, tail to tail, tail to head and head to tail.

72. The method, cell, cell line or kit of any one of claims 70 or 71, wherein each expression plasmid comprises at least a nucleic acid encoding a gene of interest (GOI).

73. The method, cell, cell line or kit of claim 72, wherein all GOIs are identical.

74. The method, cell line, kit or isolated cell of claim 72, wherein all GOIs are different.

75. The method, cell, cell line or kit of claim 72, wherein at least one GOI is different from the others.

76. The method, cell, cell line or kit of any one of claims 74 or 75, wherein the first GOI comprises a heavy chain (HC) of an antibody and the second GOI comprises a light chain (LC) of an antibody.

77. The method, cell, cell line or kit of any one of claims 70 to 76, wherein at least one expression plasmid is bicistronic.

78. The method, cell, cell line or kit of claim 77, wherein the bicistronic expression plasmid encodes a first GOI comprising the HC of the antibody and a second GOI comprising the LC of the antibody.

79. The method, cell, cell line or kit of any one of claims 70 to 78, wherein at least one landing pad plasmid is addressable.

80. The method, cell, cell line or kit of any one of claims 70 to 79, wherein each landing pad plasmid comprises two Lox sites.

81. The method, cell, cell line or kit of claim 80, wherein the Lox sites are Lox P and Lox 511.

82. The method, cell, cell line or kit of any one of claims 70 to 81, wherein each landing pad plasmid comprises a Lox site and a Frt site.

83. The method, cell, cell line or kit of any one of claims 70 to 81, wherein each landing pad plasmid comprises one or two aat sites.

84. The method, cell, cell line or kit of any one of claims 70 to 83, wherein each landing pad plasmid is addressable.

85. The method, cell, cell line or kit of claim 84, wherein each addressable landing pad plasmid comprises a pair of addressable SSRS unique to the landing pad.

86. The method, cell, cell line or kit of claim 85, wherein at least one pair of addressable SSRS is a pair of Lox sites.

87. The method, cell, cell line or kit of claim 86, wherein at least one pair of Lox sites is Lox511 and Lox P.

88. The method, cell, cell line or kit of claim 86, wherein at least one pair of Lox sites is Loxm3 and Loxm7.

89. The method, cell, cell line or kit of any one of claims 84 to 88, comprising a first addressable landing pad plasmid comprising a Lox 511 and Lox P pair of Lox sites and a second addressable landing pad plasmid comprising a Lox m3 and Lox m7 pair of Lox sites.

90. The method, cell, cell line or kit of claim 84, wherein each addressable landing pad plasmid comprises a non-cross-compatible att site.

91. A cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 20 or SEQ ID NO: 116 or a direct homologous sequence thereof.

92. A cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a nucleotide position or a polynucleotide subsequence within SEQ ID NO: 21 or SEQ ID NO: 117 or a direct homologous sequence thereof.

93. A cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a polynucleotide subsequence overlapping or encompassing SEQ ID NO: 20 or SEQ ID NO: 116 or a direct homologous sequence thereof.

94. A cell comprising a heterologous polynucleotide sequence comprising a nucleic acid encoding a gene of interest (GOI), a selection marker, a detectable marker, or a combination thereof integrated at a specific locus in the genome of the cell, wherein the locus is a polynucleotide subsequence overlapping or encompassing SEQ ID NO: 21 or SEQ ID NO: 117 or a direct homologous sequence thereof.

95. The cell of any one of claims 91 to 94, wherein the cell is a CHO cell.

96. A cell according to any one of claims 91 to 95, wherein the orthologous sequence has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 96%, about 97%, about 98% or about 99% sequence identity to SEQ ID NO:20, 21, 116, 117 or a subsequence thereof.

97. The cell of claim 98, wherein sequence identity is determined via pairwise alignment using an implementation of the Needleman-Wunsch algorithm.

98. The cell of any one of claims 91 to 95, wherein the cell comprises two landing pad plasmids or two expression plasmids.

99. The cell of any one of claims 91 to 98, wherein the cell comprises more than two landing pad plasmids or more than two expression plasmids.

100. The cell of claim 98 or 99, wherein the two landing pad plasmids are addressable.

Images