Methods for producing human induced pluripotent stem cells by homologous recombination and integrase-mediated recombination

Abstract

A method for producing human induced pluripotent stem cells from human somatic cells is provided, comprising the steps of homologous recombination and an integrase-mediated reaction.

Description

[Technical field]

[0001] The present disclosure provides a method for producing human induced pluripotent stem cells (hiPSCs) from human somatic cells by a combination of homologous recombination and integrase-mediated reactions. [Background technology]

[0002] Current methods for producing human induced pluripotent stem cells (hiPSCs) from somatic cells rely mostly on retrovirus- or lentivirus-mediated delivery of transgenes encoding a series of transcription factors, such as the Yamanaka factors (OCT3 / 4, SOX2, KLF4, and c-MYC, "OSKM") (Non-Patent Document 1) or Thomson factors (OCT3 / 4, SOX2, NANOG, and LIN28) (Non-Patent Document 2).

[0003] The problem with lentivirus- or retrovirus-mediated delivery of transcription factors is that the introduced viral vector and transgene are randomly and stably integrated into the human genome, and there is a risk of multiple viral copies being inserted into the human genome. These viral insertions result in significant genome modifications, which may lead to undesired activation or inactivation of host genes, thus impairing normal cell function or increasing the risk of tumorigenicity. Although transgenes are silenced by de novo DNA methylation during the iPSC generation process, they may be spontaneously reactivated during cell culture and differentiation. In addition, various transcription factors will show different expression levels in different cells or even in the same cell, resulting in low efficiency of iPSC generation. These drawbacks of lentivirus- and retrovirus-mediated random integration pose serious problems for both basic research and clinical applications.

[0004] performed site-specific reprogramming in mice by inserting a reprogramming cassette into fibroblasts and adipose stem cells by phiC31 integrase-mediated recombination at "pseudo attachment (att) sites" in the mouse genome (Non-Patent Document 3). The reprogramming genes in the resulting iPSCs were removed by Cre recombinase-mediated excision to reduce tumorigenicity. In this paper, the authors did not prove that this method is feasible in human cells. This method has several drawbacks and may not be suitable for application to human cells. Specifically, a "pseudo att site" is a sequence that naturally occurs in the recipient's genome and shares a certain sequence identity with the authentic att site of phiC31 integrase, so that this site can be recognized by integrase to achieve integrase-mediated recombination without the additional step of prior integration of the attachment site in the recipient's genome. However, the human genome is large, and there may be multiple sequences in the genome that can function as pseudo att sites, some of which may be located at intragenic sites. Integration at intragenic sites leads to loss of gene function. Multiple integration sites within the genome reduce the accuracy compared to integration via true att sites, and integration via pseudo att sites can lead to deletions or chromosomal rearrangements near the integration site. There remains a need to develop methods for producing iPSCs from human somatic cells that address the above-mentioned disadvantages. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takahashi and Yamanaka, 2006 [Non-Patent Document 2] Yu and Thomson, 2007 [Non-Patent Document 3] M. Karow et al., Stem Cells. 2011 November; 29(11): 1696-1704 Summary of the Invention

[0006] The present inventors have developed a methodology for producing iPSCs from human somatic cells, which includes the steps of introducing an integrase attP site together with a set of reprogramming genes into the safe harbor site by allowing homologous recombination between a donor plasmid and a safe harbor site in the genome of the somatic cell, and removing the reprogramming genes together with the plasmid sequence from the iPSCs or replacing them with a gene(s) of interest by an integrase-mediated reaction after the generation of the iPSCs, thus completing the present invention. The method of the present invention introduces the elements required for homologous recombination, reprogramming, and integrase-mediated recombination in one step by one nucleic acid construct.

[0007] Thus, in a first aspect, the present disclosure provides a 5' to 3' sequence: i. a 5' homology arm that is homologous to a first region of the locus; ii. the first attachment site of integrase I; iii. a reprogramming cassette comprising coding sequences for a set of reprogramming factors; iv. the first attachment site of integrase II; v. a 3' homology arm that is homologous to a second region of the locus; and wherein the locus is a safe harbor locus in the human genome.

[0008] In a second aspect, the present disclosure provides a vector, such as a plasmid vector or composition, comprising the nucleic acid construct of the first aspect. The vector of the second aspect (a donor vector for homologous recombination) can be used to introduce a reprogramming cassette flanked by first attachment sites for integrase into a safe harbor locus in the genome of a human somatic cell.

[0009] In a third aspect, the present disclosure provides a second vector comprising a second attachment site for integrase I and a second attachment site for integrase II, and optionally a gene of interest cassette between the two attachment sites. The second vector (a donor vector for integrase-mediated recombination) can be used to remove the inserted reprogramming cassette or to replace the inserted reprogramming cassette with a gene of interest via an integrase-mediated reaction in the presence of the corresponding integrase.

[0010] In a fourth aspect, the present disclosure provides a kit comprising a vector of the second aspect, a second vector of the third aspect, and one or two integrase expression vectors comprising a coding sequence for an integrase.

[0011] In a fifth aspect, the present disclosure provides a method for producing induced pluripotent stem cells (iPSCs) from human somatic cells, comprising: a) introducing the vector of the first aspect into a human somatic cell; b) culturing a human somatic cell under conditions that promote recombination between the 5' homology arm and the first region and between the 3' homology arm and the second region, resulting in insertion of the reprogramming cassette at the locus and generating a master somatic cell; c) culturing the master somatic cells generated by step b) under conditions promoting reprogramming to allow the generation of master iPSCs; d) optionally introducing a second vector and an integrase expression vector into the master iPSCs generated by step c); wherein the second vector comprises a second attachment site for integrase I and a second attachment site for integrase II, and optionally a gene of interest cassette between the two attachment sites, and the integrase expression vector comprises a coding sequence for integrase I and a coding sequence for integrase II; e) optionally culturing the iPSCs generated by step d) under conditions that promote integrase-mediated recombination between the first and second attachment sites of integrase I and between the first and second attachment sites of integrase II, resulting in excision of the reprogramming cassette from the genome of the iPSCs and generating iPSCs that do not contain the reprogramming cassette and, optionally, contain the gene of interest; The present invention provides a method comprising:

[0012] In a sixth aspect, the present disclosure provides a population of iPSCs produced by the method of the fifth aspect.

[0013] In a seventh aspect, the present disclosure provides the use of any of the vectors in the preparation of iPSCs.

[0014] The present invention has several advantages over currently available reprogramming systems, specifically:

[0015] Because homologous recombination occurs at safe harbor sites, the risk of gene disruption caused by random integration of reprogramming genes is greatly reduced. Integration of reprogramming genes is precise, since not only can the location of integration be predetermined, but the generated iPSCs will have only one copy of each gene integrated into their genome, and therefore reprogramming factors will be expressed at similar levels in all iPSCs.

[0016] The reprogramming genes and plasmid material can be easily removed after reprogramming by transient expression of integrase. The ability to replace the removed reprogramming gene with a gene(s) of interest during an integrase-mediated reaction opens up many potential applications.

[0017] This method can be used to reprogram a wide variety of cells, including blood cells such as PBMCs, which are known to be a challenging task due to the heterogeneity of blood cell populations and difficult cell expansion. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic showing the generation of iPSCs from somatic cells by introducing reprogramming factors via integrase-mediated recombination. [Diagram 2] FIG. 1 shows GFP expression indicating successful transfection of the plasmid. [Diagram 3] FIG. 1 shows colony morphology. [Figure 4] FIG. 1 shows immunocytochemical staining of pluripotency markers of iPSC colonies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Unless specifically defined elsewhere herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] As used in this specification, including the appended claims, the singular forms of words such as "a", "an", and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0021] In the context of this disclosure, unless otherwise indicated, the term "comprise" and its variants such as "comprises" and "comprising" are to be interpreted as meaning the inclusion of the specified elements, e.g., amino acid sequences, nucleotide sequences, properties, steps, or groups thereof, but without excluding any other elements, e.g., amino acid sequences, nucleotide sequences, properties, and steps. As used herein, the term "comprise" or any variant thereof can be replaced with the terms "contain," "include," or sometimes "have," or equivalent variants thereof. In certain embodiments, the term "comprise" also includes the scenario of "consisting of."

[0022] As used herein, "iPSC" refers to pluripotent stem cells (PSCs) generated by reprogramming of somatic cells. PSCs, including iPSCs and ESCs (embryonic stem cells), are characterized by their ability to self-renew and differentiate into any type of cell.

[0023] "Somatic cell" refers to a cell of an organism other than a germ cell.

[0024] As used herein, "master somatic cell" or "master iPSC" refers to a somatic cell or iPSC, respectively, into which a reprogramming cassette has been integrated.

[0025] "PMBC" is an abbreviation for peripheral blood mononuclear cells.

[0026] As used herein, a "cassette" or "expression cassette" refers to a DNA component comprised in a vector (e.g., a plasmid vector) and consisting of one or more genes (e.g., genes for reprogramming factors) and regulatory sequence(s) that are expressed in a host cell transfected by the vector.

[0027] "Reprogramming," as used herein, refers to the process of reverting somatic cells back into induced pluripotent stem cells.

[0028] "Reprogramming cassette" as described herein refers to an expression cassette that contains a nucleotide sequence that encodes one or more reprogramming factors. The genes of the reprogramming factors are called "reprogramming genes". A "linker sequence" is present between any two consecutive reprogramming genes. The reprogramming cassette may further include a reporter gene, a selection marker, etc.

[0029] "Donor plasmid" or "donor" refers to a plasmid carrying a sequence to be introduced into the genome via recombination. In the homologous recombination step of the method, the donor plasmid contains a nucleic acid that includes the reprogramming cassette. In integrase-mediated recombination, the donor plasmid contains a nucleic acid that includes the gene(s) of interest. In some cases, the donor plasmid in the context of this application specifically refers to a donor plasmid for homologous recombination that introduces a reprogramming cassette into the human genome, whereas the donor plasmid for integrase-mediated recombination is referred to as a "gene of interest (GOI) plasmid."

[0030] "Target genome" refers to the genome that is intended to be modified, particularly by introducing an exogenous sequence via homologous or integrase-mediated recombination.

[0031] As used herein, a "safe harbor site" or "SHS" refers to an intergenic or intragenic locus that is transcriptionally active in the target genome. "Safe" means that modifications, particularly insertions, at such sites are unlikely to disrupt normal gene function or alter the host phenotype.

[0032] "Homologous recombination" refers to the exchange of strands between two DNA molecules based on sequence homology. Homologous recombination occurs naturally in cells and plays an important role in repairing DNA double-strand breaks.

[0033] "Integrase" refers to a phage integrase that effects recombination between attachment (att) sites in the bacterial genome and the phage genome. The att site in the bacterial genome is known as attB, and the att site in the phage genome is known as attP. Integration usually requires a phage integrase in association with bacterial host factors. Certain integrases, such as phiC31 and Bxb1, function independently of host factors. Thus, they can function in cellular environments different from their native bacterial environment, making them powerful tools for site-specific recombination in cells ranging from bacteria to mammals.

[0034] "att site" refers to the attachment site for the subject integrase. When referring to integrase in this application, the att site is used interchangeably with the term "recognition site" or sometimes "recombination site".

[0035] "Gene of interest" or "GOI" refers to a gene under consideration. In the context of the methods of the present invention, GOI specifically refers to a gene that is introduced into the genome of an iPSC via an integrase-mediated reaction.

[0036] "Site-specific integration" means that the foreign nucleic acid is integrated into a predetermined location within the host genome, rather than at random sites. In the context of this application, both homologous recombination and integrase-mediated recombination result in site-specific integration of the integrated gene.

[0037] Homologous recombination As shown in Figure 1, the method of the present application includes two steps of essential recombination reactions. The first recombination reaction is a homologous recombination between a region in the genomic sequence of a somatic cell and a donor vector carrying a reprogramming gene, which introduces the reprogramming gene into the genome without disrupting normal gene function, resulting in the creation of the master somatic cell of the present application. After the first step of recombination, the master somatic cell can be reprogrammed to become a master iPSC. Then, a second step of integrase-mediated recombination is performed between the master iPSC and an "empty" construct or a donor vector carrying a gene of interest (GOI) to remove the reprogramming gene or replace it with the GOI, resulting in an iPSC that does not contain any exogenous programming gene and may further contain one or more genes of interest.

[0038] In the context of the present application, homologous recombination may be achieved by any method known in the art or developed in the future, so long as the above-mentioned result, i.e., introduction of a reprogramming gene at a desired location in the genome, e.g., a safe harbor site, can be achieved.

[0039] In one embodiment, the homologous recombination can be spontaneous homologous recombination. "Spontaneous homologous recombination" means that recombination occurs spontaneously in cells without the aid of any artificially introduced foreign enzyme.When the homologous recombination is spontaneous homologous recombination, the homologous arm contained in the donor plasmid is designed based on the sequence of the region containing the insertion position.

[0040] Studies have shown that DNA double-strand breaks (DSBs) can increase the efficiency of homologous recombination, and therefore it may be desirable to generate DSBs at the targeted site, for example by nucleases.

[0041] Thus, in some embodiments, the homologous recombination of the present application can be mediated by nucleases such as TALENs, ZFNs, or Cas9.

[0042] For example, the homologous recombination can be TALEN-assisted recombination. "TALEN" refers to transcription activator-like (TAL) effector nucleases, which can be used to perform gene editing by creating double-stranded breaks. TALENs contain a TAL effector DNA-binding domain and a DNA-cleavage domain. The DNA-binding domain contains 33-35 amino acids and can be engineered to target and bind to a specific DNA sequence as desired, and the DNA sequence can be cleaved by the DNA-cleavage domain of the TALEN.

[0043] When homologous recombination is TALEN-assisted recombination, the homology arms contained in the donor plasmid of homologous recombination are designed based on the sequence of the region where DSB occurs at safe harbor site.In addition to the donor plasmid containing reprogramming gene, an additional plasmid encoding TALEN element is also required to complete the homologous recombination of this method.TALEN element contains TAL protein that recognizes and binds to specific nucleotide and FokI nuclease that generates DSB.

[0044] For example, homologous recombination can be ZFN-assisted recombination. "ZFN" refers to zinc finger nuclease, which comprises zinc finger DNA binding domain and DNA cleavage domain.Similar to TALEN, ZFN can be designed to target specific DNA sequence as desired.

[0045] For example, homologous recombination can be carried out by CRISPR / Cas system, such as CRISPR / Cas9 system. As one of the most well-known gene editing tools, CRISPR / Cas9 includes an editable single guide RNA (sgRNA) sequence and an endonuclease Cas9 (CRISPR associated protein 9). Site-specific editing is achieved by manipulating the sgRNA. The present application also contemplates the use of other recently discovered CRISPR-associated endonucleases, such as Cas12a, Cas13a, etc.

[0046] The homologous recombination of the present application occurs in vitro, preferably under conditions that promote homologous recombination to occur.

[0047] Homologous recombination - donor plasmid The donor plasmid involved in homologous recombination contains a cassette of reprogramming genes.

[0048] The reprogramming cassette comprises a nucleotide sequence that encodes the combination of transcription factors required to achieve reprogramming. The combination of transcription factors can be any known in the art or that will prove effective in the future.

[0049] Non-limiting examples of reprogramming factors include POU5F1 (OCT3 / 4), NANOG, SOX2, LIN28A, KLF4, MYCL, MYCN, MYC, p53 knockdown, MIR302 / 367 cluster, ESRRB, REX1, GBX2, DLX4, ZSCAN10, ZSCAN4, TBX3, GLIS1, NR5A1 / 2, RARG, BMI1, KDM2B, TET1SV40L, TGBX2, NANOGP8, SP8, PEG3, and ZIC1. The reprogramming cassette of the present application may contain one or more genes encoding one or more of the reprogramming factors described above. In a preferred embodiment, the donor plasmid or reprogramming cassette contains at least a gene encoding OCT3 / 4. In another preferred embodiment, the donor plasmid or reprogramming cassette contains at least a gene encoding OCT3 / 4 and SOX2.

[0050] In certain embodiments, the combination of transcription factors may be selected from the group consisting of: (1) Yamanaka factors, OCT3 / 4, SOX2, KLF4, and c-MYC (OSKM); (2) OCT3 / 4, SOX2, and KLF4 (OSK); (3) Thomson factors, OCT3 / 4, SOX2, NANOG, and LIN28. The genes of the reprogramming factors may be arranged in any order in the cassette. Certain orders may be preferred over others. For example, for OSKM factors, it is preferred to start with the OCT3 / 4 gene and end with the c-MYC gene. However, it should be understood by those skilled in the art that the real value of the present invention lies in the design of the reaction scheme, not in the selection and arrangement of the reprogramming factors.

[0051] The nucleotide sequence encoding the reprogramming factor may be spaced apart by a nucleotide sequence encoding a linker. In one embodiment, the linker may be an IRES (internal ribosome entry site), such as a mini-IRES, EMCV IRES (IRES from encephalomyocarditis virus) or mutant. In another embodiment, the linker may be a self-cleaving peptide, such as a 2A peptide, including but not limited to T2A, F2A, E2A, P2A, etc. The advantage of an IRES is that it does not produce any peptide fragments. However, cistrons linked by an IRES may not be expressed at comparable levels. It has been reported that genes located upstream of an IRES are expressed at significantly higher levels than genes located downstream of an IRES. Rather, viral 2A peptides, when used as linkers connecting two or more genes, can produce protein products at approximately equimolar levels. Thus, in a preferred embodiment, the nucleotide sequence of a 2A peptide is used as a linker sequence to isolate the reprogramming genes.

[0052] The genes of reprogramming factors can be transcribed polycistronically under the control of a single promoter.Exemplary promoters can include EF1α, CMV, ACTB, PGK, UbC, or CAG.However, those skilled in the art can use any conventional promoter in the reprogramming cassette.

[0053] Additionally, the reprogramming cassette may include one or more of the following elements: an origin of replication, a selection marker, and a 3' non-translated sequence. For example, the origin of replication may be an F1 ori. The selection marker may be a gene that confers resistance to certain antibiotics, such as a puromycin resistance gene. The 3' non-translated sequence may be a polyadenylation sequence (poly(A)), such as TK polyA or SV40 polyA.

[0054] The reprogramming cassette may further comprise a reporter gene that indicates successful insertion of the reprogramming cassette. The reporter gene may be a gene encoding a fluorescent protein, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or bioluminescence, such as luciferase.

[0055] The reprogramming cassette is flanked by two att sites, e.g., two attP sites or two attB sites, to facilitate recombination, since the att sites are the recognition sites for integrase. During recombination, each att site is cut in the middle, and two recombined att sequences, namely attL and attR, are formed by the att fragment from the donor and the att fragment from the target genome, respectively. The att sites can be naturally occurring att sites originally from phages, or functional variants or functional fragments thereof that can be recognized by integrase and mediate the recombination reaction. In a preferred embodiment, the att sites are longer than 16 bp to avoid matching any random sequence in the genome, which would reduce the specificity of the site. For example, the att sites can be any length ranging from 17 bp to 60 bp (any integer within this range).

[0056] The donor plasmid for homologous recombination further comprises two homology arms flanking the attP-reprogramming gene(s)-attP construct or the attB-reprogramming gene(s)-attB construct, each of the homology arms of the present application being homologous to a region of the genome such that homologous recombination is triggered.

[0057] The two homology arms can be homologous to two non-overlapping fragments of a safe harbor site, for example, two non-overlapping fragments of the H11 locus, respectively. If the insertion locus is an intragenic location of a non-essential gene, the two homology arms can be homologous to two non-overlapping fragments of the non-essential gene.

[0058] The length of the homology arms can range from about 10 bp to 10 kb or even longer, for example about 50 bp to 500 bp, about 100 bp to 400 bp, for example about 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1000 bp. The two homology arms may have similar or different lengths.

[0059] The homology arms in the donor plasmid do not have to be 100% identical to their corresponding regions in the genome for homologous recombination to occur. In some embodiments, the homology arms have at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity to the corresponding genomic region, such as a safe harbor site or a fragment of a non-essential gene. In a preferred embodiment, each of the homology arms has at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to the corresponding genomic region, such as a safe harbor site or a fragment of a non-essential gene.

[0060] Depending on the mechanism of recombination, e.g. spontaneous or nuclease-mediated recombination, the selection of corresponding genomic regions of the homology arms from certain safe harbor sites or non-essential genes as well as the design of the specific sequences of the homology arms may depend on various rules.

[0061] For example, if nuclease-mediated recombination is used, the homology arms may be sequences suitable for use in a nuclease-mediated recombination system.

[0062] The two regions in the genome corresponding to the two arms of homology may be contiguous segments of each other or may be spaced apart by one or more nucleotides.

[0063] Homologous recombination - insertion locus In this method, the insertion of the reprogramming cassette into the genome does not affect the normal function of the cell, which can be achieved by inserting the foreign sequence into a locus in the genome known as a "safe harbor."

[0064] In some embodiments, the insertion locus is an intergenic safe harbor site.The safe harbor site in the human genome can be any of those known in the art, such as those described in Stefan Pellenz et al. ("New Human Chromosomal Sites with 'Safe Harbor' Potential for Targeted Transgene Insertion", HUMAN GENE THERAPY, Vol. 30, No. 7, 2019).

[0065] It has been suggested that the efficiency of recombination depends on the choice of safe harbor site. The location of the safe harbor site may also affect the continuous expression of the transgene inserted therein. Certain safe harbor sites perform better in maintaining transgene expression compared to other sites. In the present application, the safe harbor site is the location where the reprogramming factor is inserted. Both high recombination efficiency and more stable expression of the reprogramming factor are desirable for the present application. In a preferred embodiment, the safe harbor site in the human genome of the present application can maintain the continuous expression of the transgene. In a preferred embodiment, homologous recombination at the safe harbor site can result in high efficiency of recombination.

[0066] Particularly useful safe harbor sites in the present methods include, but are not limited to, H11 (Hipp11), AAVS1, hROSA26, and CCR5. More preferably, the safe harbor site in the human genome is the H11 site.

[0067] In some embodiments, the safe harbor site may be an intragenic region of a non-essential gene. By "non-essential gene" is meant that the loss of this gene's function does not significantly impair the normal function of the target cell. For example, the insertion locus may be within a gene such as TRAC, TRBC1, TRBC2, etc.

[0068] After the insertion locus is determined, the homology arms used to mediate homologous recombination can be designed based on the sequences of the regions surrounding the locus.

[0069] Homologous recombination - introduction of donor plasmid The method of the present application involves inserting a reprogramming cassette of the present application into the genome of a somatic cell via homologous recombination.

[0070] The introduction of the donor plasmid can be carried out by any conventional means in the art. For example, the donor plasmid containing the reprogramming gene can be transfected into somatic cells by various methods, including biological, chemical, and physical methods. An exemplary biological transfection is transfection mediated by viruses such as adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), etc. Examples of chemical transfection are calcium phosphate transfection, calcium chloride transfection, and transfection using cationic polymers or cationic lipids. Examples of physical transfection include direct injection, biolistic particle bombardment, and electroporation. Those skilled in the art can determine the method of transfection.

[0071] Homologous recombination - culture conditions Homologous recombination can be spontaneous homologous recombination or nuclease-assisted homologous recombination that occurs in cells cultured in vitro. Suitable cell culture conditions are known to those skilled in the art.

[0072] For the homologous recombination of the present application, there is no particular limitation on the cell culture conditions, as long as the culture conditions can support the growth of cells. Usually, the higher the recombination efficiency, the better, but one reason is that the efficiency of homologous recombination is not important in the whole methodology. At a later stage, cells will be identified and selected. Only cells that have successfully inserted reprogramming factors can be reprogrammed to become pluripotent stem cells.

[0073] Appropriate culture conditions can be determined based on the type of cells. Any somatic cells can be used as the starting point of the method, into which the reprogramming plasmid is introduced. For example, the somatic cells can be fibroblasts, which are commonly used for reprogramming. In another embodiment, the starting somatic cells are blood cells that have not been conventionally used for reprogramming. Blood cells suitable for the method, such as peripheral blood mononuclear cells (PBMCs) or CD34 + Hematopoietic stem and progenitor cells can be isolated from bone marrow, umbilical cord blood, or peripheral blood.

[0074] Exemplary media for culturing blood cells include, but are not limited to, Iscove's Modified Dulbecco's Medium (IMDM) supplemented with fetal bovine serum (FBS) or human plasma, or RPMI 1640 medium supplemented with fetal bovine serum (FBS) or human plasma, AIM V serum-free medium (Gibco), MarrowMAX™ Bone Marrow Medium (Gibco), Blood Cell Growth Medium (Sigma-Aldrich), or StemSpan™ serum-free growth medium (STEMCELL Technologies). The culture medium can be supplemented with one or more growth factors and cytokines that support the growth of blood cells. CD34 isolated from human PBMCs can be used to culture blood cells. + With respect to cells, the isolated cells can be cultured in medium supplemented with one or more cytokines or growth factors, preferably all of hSCF, hTPO, hIL-3, hIL-6, and hFlt3L, such as StemSpan™ Serum-Free Growth Medium II (STEMCELL Technologies) or StemPro™-34 SFM (Gibco).

[0075] After introduction of the donor plasmid, the cells can be maintained under conditions suitable for somatic cells, for example for a period of 2 to 10 days, 3 to 8 days, or 4 to 6 days, and then switched to conditions suitable for inducing reprogramming.

[0076] Homologous recombination - verification To select master somatic cells, which are cells into which the donor plasmid has been introduced, the cells can be cultured under selection stress if the donor plasmid contains a selection marker.

[0077] Successful recombination can be verified by conventional means in the art. For example, successful introduction of the construct of homology arm-attP-reprogramming gene(s)-attP-homology arm or homology arm-attB-reprogramming gene(s)-attB-homology arm into somatic cells can be confirmed by PCR, sequencing, and / or Southern blot. In another embodiment, in the case of a donor plasmid or reprogramming cassette that contains a reporter gene, successful introduction of the construct can be verified based on the presence of the reporter gene product.

[0078] However, validation is not required until the reprogramming process is complete after introduction of the donor plasmid: validation assays can be postponed to a later stage after somatic cells have been cultured under reprogramming conditions for an appropriate period of time.

[0079] Reprogramming After the introduction of the reprogramming cassette, the master somatic cell can be cultured under conditions that induce reprogramming to produce a master iPSC. The reprogramming conditions can include the use of a culture medium that helps induce cell reprogramming. Such a medium can include certain compounds and / or nutrients that promote reprogramming or enhance the efficiency of reprogramming. Such compounds include, but are not limited to, cytokines, growth factors, minerals, etc. The nutrients include, but are not limited to, essential and non-essential amino acids.

[0080] An exemplary medium suitable for the reprogramming process includes DMEM / F12 as a basal medium and further includes supplements such as serum supplement, amino acid supplement, minerals, etc. The supplements can be commercially available premixes, such as N-2 supplement, B-27™ supplement, GlutaMAX, or can be prepared on-site. Specific examples of media can be DMEM / F12 supplemented with 10% KnockOut serum replacement (KSR), MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol, and 100 ng / mL basic FGF, or DMEM / F12 supplemented with 1% N-2 supplement, 2% B-27™ supplement, MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol, and 100 ng / mL basic FGF, or mTeSR™ Plus medium (Stemcell).

[0081] With or without verification of successful introduction of the reprogramming cassette, the cells can be switched to reprogramming culture conditions and cultured under reprogramming conditions for a suitable period of time, e.g., at least 48 hours. In some embodiments, the cells are cultured in reprogramming medium for 14 to 21 days.

[0082] The cells are cultured in conditions suitable for somatic cells within 48 hours after introduction of the donor plasmid. An equal volume of reprogramming medium (same as iPSC culture medium) is then added to the somatic cells to achieve a 1:1 ratio of somatic cell culture medium to reprogramming medium. After 48 hours, complete medium is removed from the somatic cells, and only reprogramming medium is used for all further cultures.

[0083] Reprogramming - Verification iPSC clones can only be generated after successful homologous recombination. iPSC clones can be cultured in the same medium used for reprogramming or in various media suitable for iPSCs. Specific media for iPSCs can be DMEM / F12 supplemented with 10% KnockOut serum replacement (KSR), MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol, and 100ng / mL basic FGF, or DMEM / F12 supplemented with 1% N-2 supplement, 2% B-27™ supplement, MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol, and 100ng / mL basic FGF, or Essential 8™ medium (Gibco). iPSC clones into which the reprogramming cassette has been successfully introduced are called "master iPSCs" and can be verified by conventional means in the art, including but not limited to PCR and sequencing, or Southern blot.

[0084] In the case of verification by PCR and sequencing, gDNA can be extracted from iPSC clones. For example, multiple primer pairs can be specifically designed as follows: Pair 1: Forward primer: 20 bp sequence 300 bp outside the 5' arm of the homology arm, Reverse primer: sequence in the OCT3 / 4 gene of the reprogramming cassette, Pair 2: Forward primer: sequence in the c-MYC gene of the reprogramming cassette, Reverse primer: 20 bp sequence 300 bp outside the 3' arm of the homology arm, Pair 3: Forward primer: 20 bp sequence 300 bp outside the 5' arm of the homology arm, Reverse primer: 20 bp sequence 300 bp outside the 3' arm of the homology arm. The PCR products are subjected to electrophoresis. In the correct single allele targeting clone, a band of about 600 bp will be generated in the PCR reaction using primer pair 3, and a band of about 2 kb will be generated in the PCR reaction using primer pair 1 and primer pair 2. Further confirmation can be performed by sequencing to verify the absence of unwanted mutations in the selected clones.

[0085] Additionally, Southern blots can be performed to confirm the identity of the insert in the iPSC clones.

[0086] Any conventional means known in the art can be used to verify the pluripotency of the generated iPSCs. For example, the identity of the iPSCs can be verified by immunocytochemical staining for pluripotency markers and / or teratoma assays.

[0087] Integrase-Mediated Reaction - Integrase As shown in FIG. 1, the second recombination reaction involved in the method is an integrase-mediated reaction between a donor plasmid (GOI plasmid or empty plasmid) containing an integrase attachment site, e.g., attB site, and a genomic region into which a reprogramming gene has already been inserted, flanked by two corresponding integrase attachment sites, e.g., attP sites. This recombination is dependent on the presence of corresponding attachment sites in the donor plasmid and genomic region, and the presence of an integrase(s). Two attachment sites are required either on the donor plasmid or in the genomic region, since the recombination exchanges sequences between the two attachment sites. Those skilled in the art will understand that the type of attachment site in the donor plasmid for the integrase-mediated reaction and the type of attachment sites flanking the reprogramming gene are interchangeable, as long as one is attB and the other is attP.

[0088] In one embodiment, the two attachment sites can be the same attachment site that is recognized by a single integrase.In this case, only one integrase is required to complete recombination.However, the disadvantage of one integrase-mediated recombination is that the sequence from the donor plasmid can be inserted into the genome in either direction, so the probability of obtaining the correct recombination product is theoretically reduced by half.

[0089] Thus, in a preferred embodiment, the integrase-mediated recombination involves two integrases, with different attachment sites flanking the sequences to be exchanged, so that the orientation of the inserted sequence can be controlled.

[0090] The integrase of the present application is not naturally expressed in human cells.Integrase can be delivered and expressed by plasmid, which is referred to as "integrase plasmid" in the present application.In the case of two integrase system, two plasmids, each encoding one integrase, can be used in this method.In a preferred embodiment, integrase is transiently expressed in host cell, specifically in master iPSC.

[0091] Exemplary integrases can be serine recombinases (serine integrases), including, but not limited to, phiC31 integrase from bacteriophage phiC31, Bxb1 integrase from mycobacteriophage, TP901-01 recombinase from phage TP901-01, spoIVCA recombinase from Bacillus licheniformis, or R4 integrase from phage R4 of Streptomyces parvulus.

[0092] In certain embodiments, the integrase of the present application may be selected from Bxb1 integrase and phiC31 integrase.

[0093] For example, in the case of one integrase system, the exchanged sequences are flanked by the same attachment sites in the same plasmid. For example, in one integrase system, either Bxb1 integrase or phiC31 integrase can be used. For illustration, Bxb1 integrase is taken as an example. In the reprogramming plasmid, two Bxb1 attp sites flanked the reprogramming gene, whereas in the GOI plasmid, two Bxb1 attb sites flanked the GOI or any sequence that is inserted into the genome to replace the reprogramming cassette. Only one integrase plasmid is required to deliver the coding sequence of Bxb1 integrase to the host cell.

[0094] Another example is a two-integrase system, which is more preferred in this application. In the two-integrase system, both Bxb1 integrase and phiC31 integrase are involved in integrase-mediated recombination. In the reprogramming plasmid, the Bxb1 attP site is located on one side (e.g., 5' upstream) of the reprogramming gene, and the phiC31 attP site is located on the other side (e.g., 3' downstream) of the reprogramming gene. In the GOI plasmid, the Bxb1 attB site is located on one side (e.g., 5' upstream) of the gene of interest (if present), and the phiC31 attB site is located on the other side (e.g., 3' downstream) of the gene of interest (if present). In integrase-mediated recombination, both Bxb1 and phiC31 coding sequences are delivered to the cell by either one or two plasmids.

[0095] Integrase-mediated reaction - GOI plasmid and integrase plasmid An integrase-mediated reaction includes two or more plasmids, including a donor plasmid containing an attachment site, e.g., an attB site, and one or two integrase plasmids carrying the gene encoding the integrase(s).

[0096] The donor plasmid for the integrase-mediated reaction contains two attachment sites, e.g., attB sites, that correspond to attachment sites, e.g., attP sites, contained in the donor plasmid for the homologous recombination reaction.

[0097] To simply remove reprogramming gene from the genome of iPSC, there is no need to have a sequence between two attachment sites, such as attB sites.In one embodiment, this sequence can be the original sequence between two regions corresponding to two homology arms in genome.Then, integrase-mediated reaction will restore the original sequence of the insertion locus.

[0098] To replace the reprogramming gene with a gene of interest (GOI), the sequence between the two attachment sites, e.g., attB sites, in the donor plasmid in this step is the sequence of the GOI. In this application, the GOI can be any gene of interest. For example, the GOI can be a disease-related gene, a mutant gene, a reporter gene, a development-related gene, etc.

[0099] Integrase-mediated reaction - reaction conditions After dissociating the master iPSC into single cells, it can be subjected to further processing. Dissociation can be completed by physical or chemical methods, for example, using enzymes such as Accutase. Also, the master iPSC can be cultured under the conditions described above in the section entitled "Reprogramming", supplemented with chemicals that enhance the survival rate of iPSC as single cells, for example, ROCK inhibitor Y-27632, thiazovivin, etc.

[0100] The plasmid carrying the GOI and the integrase can be introduced via transfection means routine in the art as described above.

[0101] Integrase-mediated reaction - validation The integrase-mediated reaction aims to remove the reprogramming genes from the genome of the iPSCs, therefore the completion of this step can be verified by observing the loss of the reporter gene or by PCR reaction of the targeted region.

[0102] Applications of iPSCs The iPSCs generated by this method have numerous applications in scientific research and pharmaceutical development.

[0103] For example, if reprogramming genes are removed from iPSCs by integrase-mediated recombination, the resulting iPSCs without any exogenous genes can be used anywhere iPSCs are needed.

[0104] In some embodiments, the reprogramming gene is replaced with a gene of interest by integrase-mediated recombination, which expands the application of iPSCs to infinite possibilities. For example, iPSCs with GOIs introduced can be used to study gene function, gene mutation, and interactions in differentiation, etc. For example, the introduced GOI can be a disease-related gene, so the generated iPSCs can be used to treat diseases, for example, by gene therapy. Potential applications also include drug screening, in vitro disease modeling, cell therapy, etc. EXAMPLES

[0105] In the following section, an exemplary experimental protocol is presented.

[0106] In one exemplary embodiment, the present application can be carried out as follows and reference can be made to FIG.

[0107] The following constructs: a) A donor plasmid containing 5'H11 homology arms-phiC31 attP-genes for reprogramming factors-GFP gene-Bxb1 attP-3'H11 homology arms; b) GOI plasmid containing phiC31 attB-gene of interest-Bxb1 attB; c) an integrase expression plasmid comprising the coding sequence for phiC31 integrase and the coding sequence for Bxb1 integrase; was prepared by molecular cloning.

[0108] Human somatic cells were cultured in appropriate media prior to use.

[0109] Donor plasmids were delivered into human somatic cells using an electroporator or nucleofector, which was designated day 1. The transfected somatic cells were plated either on mouse embryonic fibroblast (MEF) feeder cells or on Matrigel / fibronectin-coated cell culture plates and cultured in the appropriate medium for 2 days. On day 3, the same volume of iPSC culture medium was added to the cells for 2 days.

[0110] The medium was switched to human iPSC culture medium on day 5. The medium was replaced with fresh human iPSC culture medium every day, and cell culture was continued until the desired morphology of human iPSC colonies could be observed.

[0111] Individual human iPSC colonies were picked. Each individual colony was cultured and expanded separately. The picked human iPSCs were characterized by one or more methods selected from immunocytochemistry for pluripotency markers, DNA methylation analysis for the OCT3 / 4 promoter, in vitro differentiation into three germ layers, teratoma assay, Southern blot for integration copy number assay, and DNA sequencing to confirm that the reprogramming factors from the donor plasmid were inserted into the H11 locus, and several candidate clones were identified.

[0112] The phiC31 integrase and Bxb1 integrase expression plasmids and the gene of interest plasmid were delivered into candidate human iPSCs to allow integrase-mediated recombination between the attP site in the H11 locus and the attB site in the gene of interest expression plasmid with the help of phiC31 integrase and Bxb1 integrase, resulting in the deletion of reprogramming factors from the donor plasmid from the H11 locus while simultaneously inserting the gene of interest into the H11 locus for subsequent use.

[0113] The same assays as above were performed to identify the final clones.

[0114] Validating successful introduction of reprogramming cassettes iPSC clones that had undergone successful homologous recombination were verified by (1) PCR and sequencing, and (2) Southern blot:

[0115] 1. PCR and Sequencing

[0116] Extract gDNA from iPSC clones.

[0117] Perform PCR using the extracted gDNA as a template with the following three primer pairs: Pair 1: Forward primer: 20 bp sequence 300 bp outside the 5' arm of the homology arm; Reverse primer: sequence within the reprogramming cassette OCT3 / 4 gene; Pair 2: Forward primer: sequence within the reprogramming cassette c-MYC gene; Reverse primer: 20 bp sequence 300 bp outside the 3' arm of the homology arm; Pair 3: Forward primer: 20 bp sequence 300 bp outside the 5' arm of the homology arm; Reverse primer: 20 bp sequence 300 bp outside the 3' arm of the homology arm.

[0118] The PCR products are run on an agarose gel. Correct single allele targeted clones will produce a band of approximately 600 bp in the PCR reaction with primer pair 3 and a band of approximately 2 kb in the PCR reaction with primer pair 1 and primer pair 2. These clones are selected for sequencing in the fourth step.

[0119] A 2 kb band is purified from a PCR reaction using primer pair 1 and primer pair 2 from the clones selected in step 3 and sequenced to identify clones that do not carry the mutation.

[0120] Clones into which the reprogramming cassette was correctly introduced were used for Southern blot analysis.

[0121] 2. Southern Blot

[0122] Extract gDNA from iPSC clones.

[0123] The DNA is digested into fragments using restriction enzymes and the DNA fragments are separated on an agarose gel.

[0124] The DNA fragments are transferred from the gel to a nylon membrane, and the nylon membrane is washed with a prehybridization solution containing salmon sperm DNA to block nonspecific DNA interactions and reduce background noise.

[0125] providing a probe targeting a promoter region within the construct; 32 Label with Pα-labeled dCTP.

[0126] The blot is incubated with a labeled probe and exposed to film to detect the probe and the DNA sequence of interest.

[0127] Only iPSC clones with one band in the Southern blot were correctly targeted clones and were used for further characterization.

[0128] Reprogramming Reprogramming was performed according to the following protocol.

[0129] CD34 from human PBMCs + Isolate the cells.

[0130] CD34 + Cells are plated in 24-well plates and cultured in StemSpan™ serum-free Growth Medium II supplemented with hSCF, hTPO, hIL-3, hIL-6, and hFlt3L.

[0131] After 3 days, cells are transfected with the reprogramming plasmid using Etta's X-Porator H1 system or Lonza's 4D-Nucleofector. Transfected cells are plated on Corning's matrix-coated culture dishes and incubated in StemSpan medium containing cytokines.

[0132] Two days after transfection, the transfected cells are fed with an equal volume of mTeSR™ Plus iPSC culture medium and cultured for an additional two days.

[0133] Four days after transfection, the medium is replaced with mTeSR™ Plus medium and the culture vessels are monitored for the appearance of iPSC colonies. Spent medium is replaced every other day.

[0134] The plasmid construct carried a neomycin resistance gene and a GFP gene for selection and screening. After transfection, GFP expression provided an indication of the level of gene expression from the locus, as shown in Figure 2. Two to three weeks after transfection, GFP + Undifferentiated iPSCs were harvested and transferred to new Corning™ matrix-coated culture dishes for expansion and passage. After three rounds of culture and passage (approximately 20 days), these colonies displayed typical hES cell morphology, as shown in FIG. 3.

[0135] Immunocytochemical staining for pluripotency markers Cells were tested for pluripotency markers by immunocytochemistry according to the following protocol: The cells expressed the pluripotency cell surface markers TRA-1-60, TRA-1-81, and SSEA-4, in addition to OCT4, SOX2, and NANOG (Figure 4).

[0136] The cells are washed briefly once with PBS and the cells are fixed with 4% paraformaldehyde in PBS for 15 minutes.

[0137] Cells are rinsed with PBS, permeabilized and blocked with 2.5% donkey serum in PBST (PBS + 0.1% TritonX-100) for 60 minutes at room temperature.

[0138] Incubate the cells for 16–18 h at 4 °C with the following primary antibodies diluted in 2.5% donkey serum in PBST: SSEA4, TRA-1-60, TRA-1-81, OCT4, SOX2, NANOG.

[0139] After washing the cells three times with PBS, the cells are incubated for 1 h at room temperature with the following secondary antibodies diluted in PBS containing 0.1% bovine serum albumin: rhodamine-labeled donkey anti-mouse IgG, rhodamine-labeled donkey anti-rabbit IgG, and rhodamine-labeled donkey anti-goat IgG.

[0140] Cells are washed three times and nuclei are stained with 1 μg / ml DAPI.

[0141] After washing three times, the staining results are observed under a fluorescent microscope.

[0142] Harvest iPSCs by treatment with 1 mg / ml ReLeSR™, centrifuge and resuspend the cells in DMEM / F12 medium.

[0143] Cells from one 60 mm dish are injected subcutaneously into non-obese diabetic / severe combined immunodeficient (NOD / SCID) mice.

[0144] A teratoma will form after 6 to 8 weeks.

[0145] After an additional two weeks, the mice are sacrificed and the tumors are autopsied.

[0146] Tumors are fixed with 4% paraformaldehyde in PBS and embedded in paraffin.

[0147] After slicing, the sections are stained with hematoxylin and eosin.

[0148] 5×10 5 Cells were seeded onto 10 cm cell culture dishes and allowed to adhere. Cells were incubated at 37°C, 5% CO2 for 48 hours.

[0149] After 2 days of culture, add 0.1 ml of colcemid, which can disrupt mitotic spindles and prevent the completion of mitosis, to each dish, mix gently, and incubate at 37°C, 5% CO2 for 2 hours.

[0150] The medium is transferred from the cell culture dish to a centrifuge tube. The dish is washed using PBS and the PBS is removed. Then 1 ml of 0.1% trypsin is added to the dish for 2 minutes at 37°C and 5% CO2. The mixture (trypsin and cells) is transferred to a centrifuge tube and mixed with the medium transferred to the centrifuge tube, after which the dish is washed using PBS. Then, the dish is centrifuged at room temperature for 10 minutes at 100 rcf.

[0151] Discard the supernatant, leaving 0.5 ml of medium and gently mix the pellet.

[0152] Resuspend the pellet in 5-7 ml of 37 °C hypotonic solution, mix thoroughly, and incubate in a 37 °C water bath for 10 min.

[0153] Centrifuge at 100 rcf for 10 minutes at room temperature. Discard the supernatant, leaving 0.5 ml of solution and gently mix the pellet. Resuspend the pellet in 5 ml of cold fixative (add drop by drop, flicking the tube between drops to prevent cell clumping).

[0154] Place the centrifuge tube on ice for at least 20 minutes. Centrifuge at room temperature for 10 minutes at 100 rcf. Discard the supernatant and add 3-5 ml of cold fixative (methanol and glacial acetic acid (3:1) freshly made and chilled before use).

[0155] The washing step is repeated three times or until the supernatant and pellet are white.

[0156] After the last centrifugation, suspend the cells in a few drops of cold fixative to obtain a slightly opaque suspension. Place 1-2 drops on a clean, damp glass slide (the slide should be rinsed with 1-2 drops of cold fixative beforehand).

[0157] Dry the slide at room temperature. Observe the chromosomes using a microscope (Olympus DP71 with 200x magnification). Count the chromosome number for approximately 50 cells. CD34 + Chromosomal G-banding analysis of PBMC-derived iPSCs revealed a normal karyotype with no evidence of clonal abnormalities.

[0158] Integrase-mediated recombination Integrase-mediated recombination was carried out according to the following protocol.

[0159] iPSCs were dissociated with Accutase into single cells and cultured at 1 × 10 for integrase-mediated reactions. 6 Collect cells.

[0160] Cells are transfected with 4 μg each of phiC31 integrase, Bxb1 integrase, and the attB donor plasmid using Etta™'s X-Porator H1 system.

[0161] The transfected cells are plated onto Corning™ matrix-coated culture dishes and the cells are cultured in mTeSR™ Plus medium supplemented with 2 μM thiazovivin.

[0162] The medium is changed daily.

[0163] One week after transfection, GFP -The undifferentiated iPSCs were harvested and expanded on new Corning™ matrix-coated culture dishes, and the researchers were able to successfully generate iPSCs in which the reprogramming genes had been removed and the gene of interest had been introduced.

Claims

1. From 5' to 3', i. a 5' homology arm that is homologous to a first region of a locus in the human genome; ii. the first attachment site of integrase I; iii. A reprogramming cassette comprising coding sequences for a set of reprogramming factors; iv. the first attachment site of integrase II; v. a 3' homology arm that is homologous to a second region of the locus; wherein said locus is a safe harbor locus in the human genome.

2. The nucleic acid construct of claim 1 , wherein the integrase I and the integrase II are integrases that are active in human cells.

3. The nucleic acid construct of claim 1 or 2, wherein the integrase I and the integrase II are the same integrase or two different integrases.

4. The nucleic acid construct of claim 2 or 3, wherein the integrase I and the integrase II are selected from phiC31 integrase and Bxb1 integrase.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the first attachment site is attP or attB, preferably attP.

6. The nucleic acid construct of any one of claims 1 to 5, wherein the safe harbor locus is the H11 locus.

7. The nucleic acid construct of any one of claims 1 to 6, wherein the 5' homology arm and the 3' homology arm have a length of about 10 bp to about 10 kb.

8. The nucleic acid construct of any one of claims 1 to 7, wherein the reprogramming cassette further comprises a selection marker and / or a reporter gene.

9. The nucleic acid construct of any one of claims 1 to 8, wherein the reprogramming cassette comprises coding sequences for OCT3 / 4, SOX2, KLF4, and c-MYC.

10. The nucleic acid construct of any one of claims 1 to 9, wherein the reprogramming cassette comprises, from 5' to 3', coding sequences for OCT3 / 4, SOX2, KLF4, and c-MYC.

11. The nucleic acid construct of any one of claims 1 to 10, wherein the coding sequences for the reprogramming factors are linked by a DNA sequence encoding a self-cleaving peptide.

12. 12. The nucleic acid construct of claim 11, wherein the self-cleaving peptide is a 2A peptide.

13. A vector comprising the nucleic acid construct according to any one of claims 1 to 12.

14. A composition comprising the vector of claim 13.

15. A kit comprising the vector of claim 13.

16. 16. The kit of claim 15, further comprising a second vector comprising a second attachment site for integrase I and a second attachment site for integrase II.

17. The kit of claim 16, wherein the second vector comprises a gene of interest (GOI) cassette located between the second attachment site of integrase I and the second attachment site of integrase II, and the GOI cassette comprises a coding sequence of the gene of interest.

18. 18. The kit of claim 17, wherein the GOI is selected from one or more of the group consisting of a therapeutic gene, a selectable marker, and a reporter gene.

19. The kit of claim 17 or 18, wherein the GOI cassette further comprises a selection marker and / or a reporter gene.

20. 20. The kit of any one of claims 16 to 19, wherein if the first attachment site is attP then the second attachment site is an attB site, and if the first attachment site is attB then the second attachment site is an attP site, preferably the second attachment site is attB.

21. The kit according to any one of claims 15 to 20, further comprising an integrase expression vector comprising the coding sequence for integrase I and the coding sequence for integrase II.

22. 1. A method for producing induced pluripotent stem cells (iPSCs) from human somatic cells, comprising: a) introducing the vector according to claim 13 into said human somatic cell; b) culturing the human somatic cell under conditions that promote recombination between the 5' homology arm and the first region and between the 3' homology arm and the second region, resulting in insertion of the reprogramming cassette at the locus and generating a master somatic cell; c) culturing the master human somatic cells generated by step b) under conditions promoting reprogramming to allow the generation of master iPSCs; A method comprising:

23. d) introducing a second vector and an integrase expression vector into the master iPSCs prepared by step c); wherein said second vector comprises a second attachment site for integrase I and a second attachment site for integrase II, and said integrase expression vector comprises a coding sequence for said integrase I and a coding sequence for said integrase II; e) culturing the iPSCs generated by step d) under conditions that promote integrase-mediated recombination between the first and second attachment sites of the integrase I and between the first and second attachment sites of the integrase II, resulting in excision of a reprogramming cassette from the genome of the iPSCs; 23. The method of claim 22, further comprising:

24. 24. The method of claim 23, wherein the second vector comprises a gene of interest (GOI) cassette located between the second attachment site of integrase I and the second attachment site of integrase II, and the GOI cassette comprises a coding sequence of the gene of interest, wherein the integrase-mediated recombination in step e) results in replacement of the reprogramming cassette with the gene of interest.

25. 25. The method of claim 24, wherein the GOI is selected from one or more of the group consisting of a therapeutic gene, a selectable marker, and a reporter gene.

26. The method of claim 24 or 25, wherein the GOI cassette further comprises a selection marker and / or a reporter gene.

27. 27. The method of any one of claims 23 to 26, wherein if the first attachment site is attP then the second attachment site is an attB site, and if the first attachment site is attB then the second attachment site is an attP site, preferably the second attachment site is attB.

28. The method according to any one of claims 22 to 27, wherein the human somatic cells are blood cells.

29. 30. The method of claim 28, wherein the blood cells are peripheral blood mononuclear cells.

30. A population of iPSCs obtainable from the method of claim 23.

31. A population of iPSCs, wherein greater than 50% of the iPSCs in the population have only one copy of the reprogramming cassette.

32. A population of iPSCs obtainable from the method of any one of claims 24 to 29.

33. A population of iPSCs, wherein more than 50% of the iPSCs in the population have only one copy of the GOI cassette.

34. A host cell comprising the vector of claim 13 or the nucleic acid construct of any one of claims 1 to 12.

35. 14. Use of the vector of claim 13 in the production of iPSCs.

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