Gene modification site

Abstract

The present invention relates to genetic engineering, particularly to a transgene insertion site, cells containing a transgene or other modification at this insertion site, vectors for targeting this insertion site, and methods for generating transgenic cells with insertion or other modification at this site. The insertion site, or "safe harbor locus," is identified within the SPATA13 gene on human chromosome 13q12.12. Mammalian cells are described that contain a genetic modification within the SPATA13 gene on chromosome 13q12.12, where the modification can be an insertion (e.g., transgene integration). Nucleic acid molecules that can induce and are configured to induce the insertion of a transgene into this insertion site are also described. Such cells or nucleic acids can be useful in therapy.

Description

[Technical field]

[0001] The present invention relates to genetic engineering, and in particular to a transgene insertion site, cells containing the transgene or other modifications at the insertion site, vectors for targeting the insertion site, and methods for generating transgenic cells with insertions or other modifications at the site. [Background technology]

[0002] The generation of transgenic cells and animals has been well established. However, the drawback of traditional transgenesis technology is that the integration of transgenes is a random phenomenon, and therefore the transgene cannot be directed to a specific location on a chromosome. Random insertion into the genome can cause problems such as insertional mutagenesis and gene silencing.

[0003] Recent techniques using site-specific recombinases, such as bacteriophage P1 recombinase Cre, and gene editing and chromosome engineering techniques using engineered endonucleases (e.g., CRISPR, zinc finger nucleases and TALENs), allow site-specific integration of transgenes into genomes. However, while site-specific insertion is now possible, many transgenic strains are still generated using traditional random insertion methods. One of the emerging challenges of such relatively new site-specific insertion techniques is the problem of the location where the transgene is specifically inserted. In addition to the need for stable integration, the main challenge is that the genomic environment of the integration site has a large effect on the expression of the transgene and can also cause downstream changes in the expression of neighboring genes, which can also cause undesired effects. The effect of chromosomal insertion location on transgene expression levels is still relatively unknown. Moreover, cells with therapeutic purposes also need to be safe and behave predictably.

[0004] A small number of sites in the human genome have been proposed as genomic safe harbors where transgenes can be inserted and expressed without significant changes in the expression of other genetic elements (Lombardo et al., 2011). However, there are not enough known and verified "safe harbor" genomic loci that can target one or more transgenes. As Papapetrou and Schambach stated (Mol Ther. 2016 Apr; 24(4): 678-684), there are no fully verified GSHs in the human genome.

[0005] Thus, there remains a need to provide transgenic cells and animals that are stable and result in acceptable expression of the transgene. For transgenic cells to have therapeutic utility, the transgenic cells must also be safe, consistent, and reproducible across batches, for example. Summary of the Invention [Means for solving the problem]

[0006] The present invention is based on the surprising identification of a genomic locus that is particularly favorable for genetic modification, e.g., for the insertion of a transgene. This is the c-MycER gene in the CTX0E03 cell line. TAM This is based on the realization that the insertion site of the transgene is safe, stable, and expressed at an effective level. Although the integration of the transgene in neural stem cells to generate the CTX0E03 cell line was a random event, it has now been realized by the inventors that a specific integration site is advantageous, which allows for the safe and stable insertion of any transgene into the cells. This has particular utility in generating cells for use in therapy.

[0007] Identification of the insertion site is beneficial because random insertion often does not work effectively and / or requires repeated attempts to achieve a stable insertion. Thus, the present invention provides a more predictable technique for genetic modification, particularly the insertion of coding sequences.

[0008] The transgenic cells of the present invention find utility in a number of applications, including as research tools, in screening, as cell therapy, for the production of biopharmaceuticals, as part of a biotechnological process, and for the recovery of microparticles, e.g., exosomes, from cells.

[0009] Inserts can also be used to provide the engineered cells with the means to produce exogenously introduced proteins or nucleic acid medicines, or to enhance the characteristics of cells to make them more suitable for use in any of the above applications, such as in biotechnological processes.For example, engineering can provide the ability to expand the growth scale of cell culture, increase the number of passages, and allow cells to stably grow in ex vivo culture, or include properties that facilitate cell tracing / labeling or purification.

[0010] The locus of the present invention provides a so-called "safe harbor" for safe and predictable manipulation. It was identified by the inventors in the CTX0E03 neural stem cell line. Thus, novel applications of the present technology are possible by introducing different or additional insertions into this locus in CTX0E03 cells, or by targeting the same locus in other (non-CTX0E03) cell types.

[0011] In a first aspect of the invention, there is provided a cell comprising a genetic modification in the SPATA13 gene, (i) the cell is not a CTX0E03 cell; and / or (ii) the genetic modification is not an insertion of a cMYC-ER transgene; Cells are provided.

[0012] In certain embodiments, the genetic modification is an insertion. In some embodiments, the genetic modification is integration of a transgene, typically stable integration of the transgene.

[0013] In various embodiments, the insertion site is: a. Within an intron of the SPATA13 gene, b. Within the third intron of the SPATA13 gene, c. Within the third intron of the cDNA clone having Genbank accession number BX648244, D. Any nucleotide between 24,083,250 and 400 bp from the P terminus on chromosome 13q12.12; e. Any nucleotide between 24,083,300 and 350 bp from the P terminus on chromosome 13q12.12 f. Any of the nucleotides between 24,083,325 and 335 bp from the P terminus on chromosome 13q12.12; or g. Between nucleotides -24,083,331 and 24,083,332 bp from the P terminus on chromosome 13q12.12 It could be.

[0014] The cell is typically a mammalian cell, more typically a human cell. When the cell is a human cell, the SPATA13 gene insertion site is present on chromosome 13q12.12.

[0015] In certain embodiments, the cell is a stem cell or a terminally differentiated cell.The cell may or may not be a neural stem cell, neural stem cell derived from fetal cortical tissue or neural stem cell line.In other embodiments, the cell may not be a neural cell and / or may not be a stem cell.

[0016] In one embodiment of the first aspect, the cell is a CTX0E03 cell that has been engineered to replace the cMycER transgene with a different transgene. The cMycER transgene may be replaced in whole or in part, or the new transgene may simply be inserted into the existing cMycER transgene (which typically disrupts the cMycER transgene and functionally replaces it with the new transgene). In some embodiments, the transgene is inserted before or after the cMyc-ER transgene, e.g., immediately before or after it, or within about 100 bp of the start or end of this transgene.

[0017] According to a second aspect, there is provided a cell of the first aspect for use in therapy.

[0018] According to a third aspect, there is provided a cell according to the first aspect for use in a biotechnological process, the process being optionally the production of stem cells, proteins or microparticles (such as exosomes).

[0019] According to a fourth aspect, there is provided a pharmaceutical composition comprising a cell according to the first aspect.

[0020] In a fifth aspect of the present invention, a method for generating a transgenic cell containing a stably integrated transgene is provided, comprising integrating the transgene into a site in the SPATA13 gene. Typically, the SPATA13 gene is present on chromosome 13q12.12. Typically, the transgenic cell is a human cell. The transgene may be or contain a human or non-human sequence. The method can be applied to the cell in vitro or in vivo. This aspect has particular utility in gene therapy, particularly when used in vivo.

[0021] The method of the fifth aspect can optionally include one or more elements of CRISPR, TALEN or other site-specific gene modification technology. In certain embodiments of the fifth aspect, the insertion site is targeted using a construct, such as a gene therapy construct, that is capable of and adapted to induce the insertion of the exogenous transgene into the insertion site after administration to a human or animal.

[0022] In a sixth aspect, there is provided a cell obtained or obtainable by the method of the fifth aspect.

[0023] In a further aspect, the present invention provides a nucleic acid molecule that can and is adapted to direct the insertion of a transgene into an insertion site, typically within the SPATA13 gene, optionally on chromosome 13q12.12, after administration to a human or animal. This nucleic acid can be used in therapy, for example gene therapy. [Brief description of the drawings]

[0024] [Figure 1-1] FIG. 1 shows the BLAST homology of two adjacent sequences (shown as Panel A and Panel B). The top line shows the prepared sequence and the bottom line shows the genomic location on chromosome 13. One mismatch is evident with adjacent sequence A due to the absence of a base call in the original sequencing. Two mismatches are evident in sequence B. These are most likely due to sequencing errors. The top line of Panel A (prepared sequence) is SEQ ID NO: 1. The bottom line of Panel A (genomic sequence) is SEQ ID NO: 2. [Figure 1-2]FIG. 1 shows the BLAST homology of two adjacent sequences (shown as Panel A and Panel B). The top line shows the prepared sequence and the bottom line shows the genomic location on chromosome 13. One mismatch is evident with adjacent sequence A due to the absence of a base call in the original sequencing. Two mismatches are evident in sequence B. These are most likely due to sequencing errors. The top line of Panel B (prepared sequence) is SEQ ID NO: 3. The bottom line of Panel B (genomic sequence) is SEQ ID NO: 4. [Figure 2-1] (A) A 1 Mb region on either side of the integration site. Distance from the chromosome 13p telomere is indicated at the top. Known genes displayed by the UCSC genome browser are shown (blue in native format). Vertical lines / rectangles represent exons and thin lines with arrows between them represent introns. Half-height rectangular exons represent non-translated exons. Superfamilies, i.e. identifiable domains / motifs, are indicated (red in native color format). The bottom row shows the distribution of the various repeat sequences. [Figure 2-2] (B) A detailed diagram of the integration site located within intron 3 of BX648244. The CpG island is also indicated in this diagram (green in the native form), as are the exons (red). The UCSC genome browser identified genes AK092754 and MGC48915 as well as BX648244 are all isoforms of the SPATA13 gene. What is particularly interesting about the integration site is that the majority of the 5' exon is untranslated, i.e., pre-messenger (displayed in the diagram as a half-height rectangle). [Diagram 3] Schematic diagram showing the region surrounding the integration site of the transgene c-myc-ERTAM (GRCh38:13:24083331-24083332) on human chromosome 13q12.12. Below, the closest genes surrounding the insertion site at the SPATA13 locus are shown with a magnification of 1M base pairs. [Figure 4]Schematic diagram showing the use of CRISPR / Cas9 to allow DNA integration in chromosome 13. (A) Representation of elements required for induction of Cas9-dependent homologous recombination in chromosome 13. (B) (GRCh38) Homologous integration of the DOI between positions chr13:24,083,331 and chr13:24,083,332. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present inventors have focused on a preferred chromosomal insertion site, which is present within the SPATA13 gene on human chromosome 13q12.12. Equivalent insertion sites at the same locus exist in the genomes of cells from other animal species.

[0026] In certain embodiments, the insertion site provides a target locus that allows the insertion of genes, gene parts or other genetic elements, for example, the target locus for gene therapy.In some embodiments, the expression of the insertion can be constitutively expressed or can be conditionally activated by using conditionally activated promoters known in the art.One such conditionally activated promoter is the TAM conditionally activated promoter used in the following examples.

[0027] In certain embodiments, one or more therapeutic genes or gene parts can be targeted to the locus by using the sequence complementary to the sequence of the target locus identified in the present invention.For example, the nucleic acid complementary to the sense strand or antisense strand of the locus of the present invention can be provided as the guide strand for gene editing technology, for example, CRISPR.

[0028] In some embodiments, this locus allows for genetic engineering of cell lines that can be used as a GMP manufacturing source for producing biologics or therapeutics. Biologics or therapeutics can include antibodies or fragments thereof, proteins, glycoproteins, peptides, lipoproteins, and the like. Some methods provide cells of the invention for the production of stem cells or microparticles. Microparticles are typically extracellular vesicles with diameters of 30-1000 nm that are released from cells. They are typically bounded by a lipid bilayer that encapsulates the biomolecule. The term "microparticle" is known in the art and encompasses a wide variety of types of microparticles, including membrane particles, membrane vesicles, microvesicles, exosome-like vesicles, exosomes, ectosome-like vesicles, ectosomes, or exovesicles. Typically, the microparticles produced by the cells of the invention are exosomes.

[0029] New sources of cells can provide advantages over existing cell lines, such as CHO and PerC6, thus providing cell lines engineered to contain exogenous coding sequences at insertion loci with stable genomes and low risk of deleterious effects on cell growth, health or toxicity.

[0030] In one embodiment, Schwann cells are immortalized by inserting a conditional oncogene into the insertion site. Thus, such cells may have utility in nerve repair and / or regeneration, typically by supporting both axonal growth and myelination. The conditional oncogene may be, for example, C-mycER or L-mycER.

[0031] The modified cells of the present invention can be used in cell therapy in humans or animals. In one embodiment, the cells of the first aspect are provided for use in treating a disease or genetic condition in a human or animal in need of such treatment. Also provided is a method of treating a patient in need thereof, comprising administering to the patient a therapeutically effective amount of (i) the transgenic cells of the present invention, or (ii) a nucleic acid vector capable of inducing the insertion of a transgene into an insertion site. The patient may also be treated by administering a guide RNA (e.g., sgRNA) targeted to the insertion site, optionally with a Cas endonuclease, e.g., Cas9. The patient is typically a human.

[0032] The process for site-specific integration may typically include 1) introducing a targeting vector containing a gene of interest into a mammalian cell, and 2) screening and selecting transfected cells that have integrated the gene of interest into a particular genomic locus. Site-specific gene insertion is known in the art, for example, as described in Nature Methods volume 10, page 13 (2013).

[0033] Also provided herein are nucleic acids, vectors and gene therapy constructs for targeting insertion sites, typically resulting in improved targeting for safer and more effective gene therapy in humans or animals.

[0034] Modification site The present invention identifies a particularly useful site in the human genome that can be used for genetic modification.This site is typically used for the insertion of the nucleic acid encoding the protein of interest, as in the case of CTX0E03 cells that identified this site, but other genetic modifications can be made at this site.This locus is generally referred to herein as the insertion site.

[0035] The insertion site was identified in the SPATA13 gene on human chromosome 13q12.12. In chimpanzees (Pan troglodytes), the SPATA13 gene is also located on chromosome 13, but this chromosomal location differs in other animals, being chromosome 14 in mice (Mus musculus), chromosome 15 in rats (Rattus Norvegicus), chromosome 17 in rhesus monkeys (Macaca mulatta), chromosome 24 in zebrafish (Danio rerio) and chromosome 2 in African clawed frogs (Xenopus laevis). In some embodiments, the SPATA13 gene described herein is targeted for modification in any animal cell, for example, within the third intron of the SPATA13 gene. Typically, the animal cell is a mammalian cell, more typically an ape, such as a chimpanzee or gorilla. More typically, the insertion site is an insertion site in a human cell.

[0036] The exact location of the insertion in CTX0E03 cells is between nucleotides 24,083,331-332 bp from the P terminus on (GRCh38) chromosome 13q12.12. However, it is expected that equivalent results can be obtained when a site in this general region, for example within 10 kb, or within 5 kb, within 2.5 kb, for example within 1000 bp or within 500 bp of this specific site, is targeted according to the present invention. In certain embodiments, the locus targeted for modification may be within an intron of the SPATA13 gene. In further embodiments, the locus is within the third intron of the SPATA13 gene. Typically, the locus is within the third intron of the cDNA clone having Genbank accession number BX648244. More specifically, the locus can be located on chromosome 13q12.12 anywhere between nucleotides 24,083,250 and 400 bp from the P terminus, anywhere between nucleotides 24,083,300 and 350 bp from the P terminus, or anywhere between nucleotides 24,083,325 and 335 bp from the P terminus.

[0037] The insertion site is referred to as "GRCh38:13:24083331-24083332." GRCh38 refers to the version of the human genome reference sequence currently used by the UCSC browser, as will be appreciated by those of skill in the art.

[0038] The insertion site was identified in a neural stem cell designated "CTX0E03" deposited by ReNeuron Limited at the European Collection of Authenticated Cell Cultures (ECACC), PortonDown, UK, and having ECACC accession number 04091601.

[0039] The cells of the CTX0E03 cell line are pluripotent cells originally derived from 12-week human fetal cortex. The isolation, production and protocols for the CTX0E03 cell line are described in detail by Sinden et al. (US Pat. No. 7,416,888 and EP 1645626). CTX0E03 cells are not "embryonic stem cells". That is, they are not pluripotent cells derived from the inner cell mass of the blastocyst, and the isolation of the original cells did not result in the destruction of the embryo. In growth medium, CTX0E03 cells are nestin positive, with a low percentage of GFAP positive cells (i.e., the population is negative for GFAP).

[0040] CTX0E03 is a clonal cell line that contains a single copy of the c-mycER transgene delivered by retroviral infection and is conditionally regulated by 4-OHT (4-hydroxytamoxifen). When integrated into the insertion site identified herein, the c-mycER transgene expresses a fusion protein that stimulates cell proliferation in the presence of 4-OHT, thus allowing for regulated proliferation when cultured in the presence of 4-OHT. The cell line is clonal, grows rapidly in culture (doubling time 50-60 hours), and has a normal human karyotype (46XY). It is genetically stable and can be propagated in large quantities. The cells are safe and non-tumorigenic. In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes. When transplanted into the ischemic brain, such cells migrate only to the damaged areas of the tissue.

[0041] Cells of the CTX0E03 cell line may be cultured under the following culture conditions. Human serum albumin 0.03% Human transferrin 5μg / ml Putrescine dihydrochloride 16.2μg / ml Human recombinant insulin 5μ / ml Progesterone 60ng / ml L-Glutamine 2mM Sodium Selenite (Selenium) 40ng / ml

[0042] Additionally, basic fibroblast growth factor (10 ng / ml), epidermal growth factor (20 ng / ml) and 100 nM 4-hydroxytamoxifen are added for cell proliferation. Cells can be differentiated by removal of 4-hydroxytamoxifen. Typically, cells are cultured in 5% CO 2 / 37°C or 5%, 4%, 3%, 2% or 1% O 2The CTX0E03 neural stem cell line can be cultured in either low or high oxygen conditions. Such cell lines do not require serum for successful culture. Serum is required for successful culture of many cell lines, but contains many contaminants. The advantage of the CTX0E03 neural stem cell line, or any other cell line that does not require serum, is that serum contamination is avoided.

[0043] Genetic modification The present invention provides for the modification of cells at the loci described herein ("insertion sites").

[0044] Typically, the modification is the insertion of a nucleic acid. The nucleic acid inserted into the insertion site can be exogenous to the cell. Alternatively, the nucleic acid inserted can be naturally found at a different locus in the cell. Typically, the nucleic acid inserted is DNA, but can also be RNA, or a hybrid containing DNA or RNA, or another nucleic acid, a non-standard nucleic acid, or a polymer thereof.

[0045] The inserted nucleic acid may be a coding or non-coding sequence. In a typical embodiment, the nucleic acid sequence is a coding sequence of DNA that causes expression of a protein of interest. The inserted nucleic acid may be from the same species as the cell to be modified (homologous insertion) or not (heterologous insertion). The inserted sequence may include one or more regulatory sequences, such as promoters, necessary to ensure that the inserted sequence is functional. In the coding sequence, the functional sequence is a sequence that can be successfully transcribed.

[0046] In some embodiments, the inserted nucleic acid is a transgene. In certain embodiments, for example, the inserted transgene may improve the expression of a recombinant protein in the cell. Examples of such transgenes include, but are not limited to, EBNA-1, GS, XBP1, or ERO-La (Hunter M, et al. 2018. Current Protocols in Protein Science). In other embodiments, the inserted transgene confers immortality or conditional immortality to the cell. In other embodiments, the cell is inserted with a new or enhanced function, such as a new cell surface receptor or a new enzymatic or structural function. Such engineered cell lines are chimeric antigen receptors engineered into immune cells, such as T cells or natural killer cells. Thus, CAR-T and CAR-NK cells can be generated by inserting chimeric antigen receptors into the insertion sites of the invention in T cells (e.g., CD8+ cytotoxic T lymphocytes) or NK cells. CAR-T and CAR-NK cells are well known for use in therapy, for example, in methods of treating cancer, such as hematological cancers.

[0047] The result of the insertion will be the production of a sequence (and hence a cell) in which the sequence has been inserted at a locus that is not naturally occurring, ie, does not exist in the unmodified chromosome.

[0048] The insertion sequence itself can be a non-naturally occurring artificial sequence, for example, produced by recombination. An example is a fusion construct of two sequences, for example, encoding a fusion protein. An example of a fusion protein is a chimeric antibody. Other non-naturally occurring proteins include humanized antibodies, which contain a mixture of non-human CDRs and human constant regions and variable framework sequences, and chimeric antigen receptors, which typically contain an antigen-binding extracellular portion derived from an antibody, and a T cell receptor transmembrane portion and an intracellular portion. CAR-T cells generated using CRISPR to target CAR coding sequences to the insertion site of the present invention are an exemplary embodiment.

[0049] In one aspect of the present invention, the cell to be modified is a mammalian cell. In some embodiments, the mammalian cell is either a human cell or an animal cell. The cell can be from muscle, epithelium, connective or nervous system. Examples of mammalian or human cells include, but are not limited to, somatic cells, neuronal cells, muscle cells, red blood cells, white blood cells, immune cells, T cells, CD4+ T cells, CD8+ T cells, B cells, bone cells, adipocytes, skin cells, cardiac cells, pancreatic cells and liver cells. In some embodiments, the cell is a stem cell, a progenitor cell, a multipotent cell, a pluripotent cell, an induced pluripotent stem (iPS) cell, or a non-stem (terminally differentiated) cell.

[0050] When used in therapy, the cells can be allogeneic or autologous to the patient. Autologous cell therapy is becoming well established, such as autologous CAR-T cell therapy.

[0051] In some embodiments, the locus allows for the engineering of cell lines that can be used as GMP manufacturing sources for the production of biologics or therapeutics. Examples of human cell lines include, but are not limited to, HeLa, HEK293 cells, U2OS, HCT116, MDA-MB-231, MDA-MB-435, U87, U251, Raji cells, Jurkat cells, PC3, MCF-7, Saos-2, HL-60, and LNCAP cells. Examples of animal (mammalian) cell lines include, but are not limited to, CHO, BHK, NS0, SP2 / 0, YB2 / 0, COS, MDCK, MSC-1, CAD, P19, NIH3T3, L929, N2a, and J558L cells. CHO cells are commonly used for the production of biopharmaceuticals and are exemplary cells for such embodiments.

[0052] Immortalization and conditional immortalization In certain embodiments, the nucleic acid inserted into the insertion site is a sequence that confers immortality to cells. For example, the nucleic acid inserted can code for an immortalization factor. Immortalization factors are well known in the art and include one or more viral oncogenes and hTERT (human telomerase reverse transcriptase) or combinations thereof. Viral oncogenes include adenovirus E1A / E1B genes, simian virus 40 large T antigen (SV40Tag) and human papillomavirus 16 (HPV16) E6 / E7 genes.

[0053] In other embodiments, the inserted sequence confers conditional immortalization to the cells, where expression of the immortalization factor can be controlled. Conditional immortalization factors are well known in the art and include temperature sensitive SV40Tag, C-MycER or L-mycER.

[0054] In conditionally immortalized cells, the expression of the immortalizing factor can be controlled without adversely affecting the generation of therapeutically effective stem cells. This can be achieved by introducing an immortalizing factor that is inactive unless the cells are provided with an activating agent. Such an immortalizing factor can be a gene such as c-mycER. The c-MycER gene product is a fusion protein that contains a c-Myc variant fused to the ligand-binding domain of a mutant estrogen receptor. c-MycER alone drives cell proliferation in the presence of the synthetic steroid 4-hydroxytamoxifen (4-OHT) (Littlewood et al. 1995). This method allows for the regulation of neural stem cell proliferation in vitro while avoiding undesirable effects on host cell proliferation in vivo (e.g., tumor formation) due to the presence of c-Myc or the gene encoding it in neural stem cell lines.

[0055] CTX0E03 is an example of a conditionally immortalized stem cell line. Exemplary conditionally immortalized cell lines include CTX0E03, STR0C05 and HPC0A07 neural stem cell lines, which have been deposited by the applicant of this patent application, ReNeuron Limited, at the European Collection of Animal Cultures (ECACC), Vaccine Research and Production Laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, and have accession numbers 04091601 (CTX0E03), 04110301 (STR0C05) and 04092302 (HPC0A07). The origin and origin of these cells are described in EP 1645626 and US Patent No. 7416888.

[0056] In one embodiment, the invention provides a cell that contains an insertion of the same conditionally immortalizing transgene, but not CTX0E03, at the same site (i.e., the insertion locus described herein). This may be a stem cell, such as a neural stem cell, mesenchymal stem cell or hematopoietic stem cell, or a non-stem cell, such as a differentiated cell. Other cells are described elsewhere herein.

[0057] An example of a differentiated cell that can be engineered to contain a c-mycER transgene at the insertion locus is a Schwann cell. Thus, for the avoidance of doubt, one aspect of the present invention provides a Schwann cell having an insertion of c-MycER (or L-Myc-ER) at the insertion site to generate a conditionally immortalized Schwann cell.

[0058] Integration site targeting Site-specific insertion of nucleic acid into genome can be achieved by genome editing tools such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) RNA-guided nucleases (Gaj T, et al. 2013. Trends in Biotechnology). These are all well-known methods in the field of genome editing. When site-specific DNA double-strand breaks (DSBs) or single-strand breaks (SSBs) are induced by engineered nucleases, the target locus is typically repaired by one of the two major DNA damage repair pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR). Those skilled in the art will understand how to use such techniques to target identified genomic loci.

[0059] Meganucleases ZFN and TALEN have been used extensively for genome editing. Meganucleases are engineered versions of naturally occurring restriction enzymes. Such enzymes typically have extended DNA recognition sequences (e.g., 14-40 bp). ZFN and TALEN are artificial fusion proteins composed of an engineered DNA binding domain fused to a non-specific nuclease domain. Zinc fingers with customized specificity and TALE repeat domains can be linked together into arrays that bind extended DNA sequences (Sander JD, and Joung K. 2014. Nature Biotechnology).

[0060] The CRISPR-Cas9 system also allows targeted editing of DNA. The system targets DNA by pairing with a guide RNA (gRNA) molecule that binds to the target DNA by base complementarity, allowing precise DNA cleavage. The gRNA is approximately 100 nt in length. The targeting specificity of the CRISPR-Cas9 system is determined by a 17-21 nucleotide sequence at the 5' end of the gRNA that is complementary to the target site. In the S. pyogenes / Streptococcus pyogenes CRISPR-Cas9 system, the desired target sequence must immediately precede a 5' PAM motif (NGG).

[0061] HDR is a desired repair pathway for inserting nucleic acid into the genome of a cell. Wild-type Cas9 enzyme can be used to introduce double-strand breaks into DNA, and mutant Cas9 enzymes that introduce single-strand breaks have been generated. In some methods, mutant Cas9 enzymes that only create single-strand breaks can be used to promote HDR (which occurs less frequently than NHEJ). These mutant Cas9 enzymes can be paired with gRNAs that target the sense and antisense strands of DNA at the insertion site to introduce targeted double-strand breaks, which are repaired by HDR in the presence of the exogenous nucleic acid to be inserted (Ran FA, et al. 2013. Cell; Koch B, et al. 2018. Nature Protocols).

[0062] Insertion of a nucleic acid into a target integration site can be achieved by designing a donor plasmid containing short homology arms, which are the insertion sites of the present invention. The homology arms are typically 100-2000 bp in length (Koch B, et al. 2018. Nature Protocols).

[0063] In some embodiments, the present invention provides gRNAs, typically isolated gRNAs, designed to hybridize to the insertion sites identified herein. The design of CRISPR guide RNAs is well known in the art. The 17-21 nt sequence at the 5' end of the gRNA that is complementary to the target site is typically at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or completely complementary to the 17-21 nt portion of the insertion site identified herein. In some embodiments, the target-complementary region of the gRNA is complementary to a region within an intron of SPATA13, typically the third intron of SPATA13.

[0064] In some embodiments, the key gRNA region is complementary to a sequence on chromosome 13q12.12.

[0065] In certain embodiments, the 17-21 nt sequence at the 5' end of the gRNA is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 99% identical, or completely complementary to a 17, 18, 19, 20, or 21 nt sequence spanning 24,083,331 to 24,083,332 bp nucleotides from the P end on human chromosome 13q12.12. In certain embodiments, the complementary region is 20 nt in length.

[0066] In some embodiments, the present invention provides compositions or kits comprising a DNA vector encoding the Streptococcus pyogenes Cas9 endonuclease and a single-stranded guide RNA as discussed above.

[0067] The insertion site was identified in human cells.In some embodiments, the insertion locus in non-human cells can be identified by hybridization of gRNA used by the CRISPR-Cas9 system of genome editing, which is designed to target the insertion site in CTX cells as discussed above.Without being bound by theory, it is understood that the gRNA designed to hybridize with the insertion site in CTX0E03 cells will hybridize with similar insertion sites in various mammalian cells.Therefore, potential insertion sites in mammalian cells can be identified by gRNA hybridizing with the DNA insertion site identified in the present invention.

[0068] Hybridization is usually performed under stringent conditions that are selected to reduce the possibility of non-complementary hybridization, which are known to those skilled in the art. Examples of suitable hybridization conditions are described in Nucleic Acid Hybridization: A Practical Approach (BD Hames and SJ Higgins, editors IRL Press, 1985). An example of stringent hybridization conditions is incubation overnight at 42°C in a solution containing 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate and 20μg / ml denatured fragmented salmon sperm DNA, followed by washing with 0.1xSSC at about 65°C.

[0069] Pharmaceutical Compositions The engineered cells and vectors and nucleic acids of the present invention are useful in therapy and can therefore be formulated as pharmaceutical compositions. Pharmaceutically acceptable compositions typically contain at least one pharma- ceutically acceptable carrier, diluent, vehicle and / or excipient in addition to the product of the present invention. An example of a suitable carrier is lactated Ringer's solution. A thorough discussion of such ingredients is provided in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0070] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0071] The composition may also include a small amount of pH buffering agent, if necessary. The composition may include a preservation medium, such as Hypothermosol®, commercially available from BioLifeSolutions Inc., USA. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions include a prophylactically or therapeutically effective amount of prophylactic or therapeutic stem cells, preferably in purified form, together with a suitable amount of carrier to provide a form for proper administration to a subject. The formulation must be adapted to the method of administration. In a preferred embodiment, the pharmaceutical composition is sterile and in a form suitable for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0072] The pharmaceutical composition of the present invention can be in various forms. These include, for example, semi-solid and liquid dosage forms, such as lyophilized formulations, frozen formulations, solutions or suspensions, injectable and infusion solutions. Preferably, the pharmaceutical composition is for injection.

[0073] The pharmaceutical composition is generally in aqueous form. The composition may contain preservatives and / or antioxidants.

[0074] To adjust the osmotic pressure, the pharmaceutical composition may contain a physiological salt, such as a sodium salt. Sodium chloride (NaCl), which may be present at 1-20 mg / ml, is preferred. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride and calcium chloride.

[0075] The composition may include one or more buffers. Typical buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer or citrate buffer. The buffer is typically included at a concentration in the range of 5-20 mM. The pH of the composition is generally 5-8, more typically 6-8, for example 6.5-7.5 or 7.0-7.8.

[0076] Preferably, the composition is sterile. Preferably, the composition is non-pyrogenic.

[0077] In an exemplary embodiment, the cells are suspended in a composition comprising one, two, three, four, five, six, seven, eight, nine, ten or more excipients selected from 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), Na+, K+, Ca2+, Mg2+, CI-, H2P04-, HEPES, lactobionic acid, sucrose, mannitol, glucose, dextran 40, adenosine and glutathione. In one embodiment, the composition comprises all of such excipients. Typically, the composition comprises a dipolar aprotic solvent, e.g., DMSO. Suitable compositions are commercially available, e.g., HypoThermasol® FRS. Such compositions are advantageous because they allow the cells to be stored for long periods (hours to days) at 4°C to 25°C or at cryothermic temperatures, i.e., temperatures below -20°C. The stem cells may then be administered in this composition after thawing.

[0078] The invention will now be further described with reference to the following non-limiting examples. EXAMPLES

[0079] A) Safe Harbor Loci for Gene Expression Introduction Insertion of transgenes for high gene expression in human cells requires the use of safe harbor loci (SHLs) that do not affect the gene expression pattern of the target cells.

[0080] So far, only three SHLs have been identified in human cells: AAVS1 on chromosome 19, CCR5 on chromosome 3, and ROSA26. Both CCR5 and AAVS1 are located in a region that contains several genes, including cancer-related genes that may be dysregulated by transgene integration. ROSA26 is also located near the gene, and there is no further testing to confirm the usefulness or safety of this locus in human cells.

[0081] Therefore, the new SHL discovery will be highly beneficial for the safe high expression of transgenes for therapeutic purposes.

[0082] Identification of SHL at 13q12.12 (GRCh38:13:24,083,331-24,083,332) Generation of the therapeutic human cell line CTX0E03 contained the recombinant gene c-myc-ER TAM This immortalization involved high expression of c-myc-ER. This was achieved by using a retrovirus that randomly inserted a single copy of a gene at a specific location on chromosome 13. We mapped this region (GRCh38:13:24,083,331-24,083,332) and placed it within an intron of the SPATA13 gene. We did not find any genes in this vicinity (<180kb) (Figures 1 and 3 and Table 2), so it is highly unlikely that any genes could be altered by this insertion or any event of chromatin remodeling due to this insertion. As c-myc-ER is still active in our CTX0E03 cell line, this clearly indicates that the chromatin conformation is in an open stage that allows for sustained expression of the transgene. None of the genes identified in the 1M base pair region surrounding the insertion region have been identified as cancer-related genes.

[0083] Therefore, the inventors believe that this specific site on chromosome 13 has the fundamental properties of SHL and can be targeted in any human cell to insert any gene for gene therapy using recombinant techniques, such as non-protein-dependent homologous recombination, TALEN nucleases or CRISPR / Cas9 nucleases.

[0084] Cas targeting of SHL For example, Cas9 can be used with a small piece of complementary RNA known as a guide RNA (sgRNA) to cleave a specific location within DNA and insert any desired DNA sequence.

[0085] To trigger this specific recombination, we use one or more sgRNAs complementary to the region adjacent to the position on GRCh38:13:24,083,331-24,083,332. The sgRNAs are delivered to the cell along with Cas9 as an expression vector or ribonucleoprotein complex (RNP) by transfection, transduction, electroporation, or any other method of delivering DNA, RNA, and / or protein. The DNA of interest (DOI), also delivered along with Cas9 and one or more sgRNAs, is flanked on both sides by a 5' arm complementary to the position of chr13:24,083,331 and a 3' arm complementary to the position of chr13:24,083,332 (Figure 4A).

[0086] Once in the nucleus, the RNP complex cleaves chromosomal DNA at the location targeted by the sgRNA, triggering DNA repair mechanisms. The DOI, which has complementary arms to chromosome 13, is recognized by the cell's recombination machinery and inserted into the complementary region.

[0087] This allows for the insertion of a DOI between positions chr13:24,083,331 and chr13:24,083,332 in any human cell line.

[0088] B) Analysis of the transgene insertion site in CTX0E03 1 Introduction CTX0E03 expresses the conditional immortalization gene cmycER TAM CTX0E03 is a neural stem cell line derived from primary embryonic neural stem cells after transduction with mycER. The transgene was engineered into the retroviral vector pLNC-X (Clontech) and cells were transduced using retrovirus generated using the packaging cell line TEFLY-A. Insertion of the transgene into the target cell genome is by random integration via the viral LTR. In the CTX0E03 cell line, we have identified the mycER site, which should be located within chromosome 13. TAMThe purpose of this report is to evaluate the possibility of post-insertion mutagenesis as a result of transgene integration.

[0089] overview Bioinformatic analysis of the insertion site and the surrounding genome indicated that the risk of oncogenic activation as a result of post-insertion mutagenesis in the CTX0E03 cell line was very low.

[0090] CTX0E03 insertion site Retroviral insertion sites were investigated for the CTX0E03 neural stem cell line, and studies included bioinformatic analysis to assess the possibility that retroviral insertions could render the cell line tumorigenic as a result of post-insertion mutagenesis.

[0091] Post-insertion mutagenesis is a well-documented phenomenon. Retroviruses insert apparently randomly into chromosomal DNA sequences, resulting in the disruption of existing DNA sequences. They therefore have the potential to mutate any gene into which they may insert. An example of this phenomenon that has raised concerns was the development of leukemia in three children during a French X-SCID gene therapy trial using retroviral vectors (see Science. 2005 Mar 11;307: 1544-5). Because the CTX0E03 cell line was generated using a retroviral vector, it is important to consider the possibility that post-insertion mutagenesis and oncogenic activation may occur.

[0092] Three hypotheses were examined in this study. 1. The provirus was inserted within a gene with oncogenic potential in a manner likely to activate this potential; 2. The provirus is inserted into or near an endogenous retrovirus, thereby causing activation or recombination; and 3. The virus was inserted close enough to a known oncogene that activation of the oncogene could result.

[0093] 2. Insertion site location – SPATA13 c-Myc-ER TAM The flanking sequences provided for allowed the placement of the insertion between nucleotides −24,083,331 and 24,083,332 (GRCh38) bp (from the P end) of chromosome 13q12.12. See FIG. 1.

[0094] Retroviruses are believed to preferentially insert into intronic sequences, and this is the case here as well. The integration site is located within the third intron of the cDNA clone with accession number BX648244. This clone represents a splice variant of the SPATA13 or spermatogenesis-associated 13 gene. The first 16 exons, including the two exons flanking the integration site, are "pre-messenger" sequences; that is, they are non-coding sequences.

[0095] The structure and function of SPATA13 were investigated by bioinformatics analysis.

[0096] 2.1 Structure SPATA13 is expressed in many tissues. Alternative splicing generates 18 different transcripts. The gene contains 8 putative alternative promoters with 37 introns. It has a very long 3'UTR. The pre-messenger contains up to 16 exons and spans 342 kb.

[0097] 2.2 Homology An amino acid homology search by BLAST (www.ncbi.nlm.nih.gov / BLAST) showed 62% homology to ARHG4 (Rho guanine nucleotide exchange factor 4 [APC-stimulated guanine nucleotide exchange factor]), which acts as a guanine nucleotide exchange factor (GEF) for RhoA and RAC1 GTPases. Binding of APC can activate RAC1 GEF activity. The APC-ARHG4 complex is thought to be involved in cell migration as well as E-cadherin-mediated cell-cell adhesion.

[0098] 2.3 Motifs (expertly edited descriptions) The complement C1q protein motif is found in three isoforms of this gene. C1q is a subunit of the C1 enzyme complex that activates the serum complement system.

[0099] The collagen triple helix repeat motif is found in six isoforms of this gene. The sequence is predominantly a GXY repeat, and the polypeptide chain forms a triple helix. The first position repeat is glycine, and the second and third positions can be any residue, but are often proline and hydroxyproline.

[0100] Pleckstrin-like motifs are found in four isoforms of this gene. The "pleckstrin homology" (PH) domain is a domain of about 100 residues that is present in a variety of proteins involved in intracellular signaling or as components of the cytoskeleton. The function of this domain is unclear, with several putative functions suggested: binding to beta / gamma subunits of heterotrimeric G proteins, binding to lipids (e.g., phosphatidylinositol-4,5-bisphosphate), binding to phosphorylated Ser / Thr residues, and binding to membranes by an unknown mechanism. Various PH domains may have quite different ligand requirements.

[0101] The OH motif is found in four isoforms of this gene. The Rho family GTPases Rho, Rae, and CDC42 regulate a wide variety of cellular processes. Like all members of the Ras superfamily, Rho proteins cycle through active GTP-bound and inactive GDP-bound conformational states. Activation of Rho proteins by release of bound GDP and subsequent binding of GTP is catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl family. Proteins encoded by members of the Dbl family share a common domain of about 200 residues (termed the Dbl homology or DH domain), which has been shown to encode specific GEF activity for many Rho family members. In addition, all family members possess a second shared domain termed the pleckstrin homology (PH) domain. The PH domain is always located immediately C-terminal to the DH domain, and this invariant localization suggests a functional interdependence between these two structural modules. Biochemical data established the role of the conserved DH domain in Rho GTPase interaction and activation, and the role of the tandem PH domain in intracellular targeting and / or regulation of DH domain function. The DH domain of Dbl has been found to mediate oligomerization, which is the most homophilic in nature. In addition to the tandem DH / PH domains, Dbl family GEFs contain diverse structural motifs such as serine / threonine kinase, RBD, PDZ, RGS, IQ, REM, Cdc25RasGEF, CH, SH2, SH3, EF, spectrin or lg. The DH domain consists of three structurally conserved regions separated by further variable regions. It does not share significant sequence homology with other subtypes of small G protein GEF motifs (e.g., Cdc25 domain and Sec7 domain, which specifically interact with Ras and ARF family small GTPases, respectively) or with other Rho protein interaction motifs, indicating that Dbl family proteins are evolutionarily unique.

[0102] The SH3 motif (src homology 3) is found in six isoforms of this gene. SH3 domains are low molecular weight protein modules that contain about 50 amino acid residues. They are found in a wide variety of intracellular or membrane-associated proteins, such as various proteins with enzymatic activity, adaptor proteins lacking catalytic sequences, and cytoskeletal proteins. Although the function of SH3 domains is not fully understood, they may mediate a wide variety of processes, such as increasing the local concentration of proteins, changing their subcellular location, and mediating the assembly of high molecular weight multiprotein complexes.

[0103] A variant SH3 motif is found in five isoforms of this gene. SH3 (Src homology 3) domains are often associated with proteins involved in signal transduction associated with cytoskeletal organization.

[0104] 2.4 Cell location NCBI Locuslink indicates functions in phosphate transport, cell adhesion and protein binding. Locuslink predicts cytoplasmic location and Psort predicts nuclear localization. Different localizations can be applied to different isoforms.

[0105] 2.5 Conclusion SPATA13 is a gene of unknown function. The structure tentatively suggests that it may be a guanine exchange factor, but this is a very preliminary conclusion since it is based only on bioinformatics. This does not a priori suggest that SPATA13 is a putative oncogene or a gene with any tumorigenic function.

[0106] We can conclude that retroviral insertion does not destroy any protein-coding sequence or any non-coding non-translated sequence. Any splice donor or acceptor sites are not destroyed as well. Therefore, we conclude that it is highly unlikely that any mutant protein can be produced from this locus or any disrupted or truncated transcript can appear. There is a possibility that RNA processing can be affected. Enhancer sequences are sometimes found to be located in intronic sequences, so they may be considered to be affected by insertion. Nevertheless, such an effect seems unlikely, since the part of the gene where insertion occurs is the 5' region dispersed between non-coding exons.

[0107] We conclude that it is highly unlikely that oncogenic activation by SPATA13 occurs as a result of this insertion event.

[0108] 3. Repetitive sequences

[0109] [Table 1]

[0110] The insertion site and the upstream and downstream chromosomal regions were analyzed to discover the presence of repeated sequences, sequences that may harbor endogenous retroviruses or transposable elements.

[0111] Only two types of transposable elements were found within the ±2.5 kb region of the integration site: long interspersed repeat elements (LINEs) and short interspersed repeat elements (SINEs).

[0112] LINEs are 6 kb long transposons that harbor an internal polymerase II promoter and encode two open reading frames (ORFs). Among LINEs, only LINE1 is active in humans. During translation, LINE RNA associates with its own encoded protein and translocates to the nucleus where an endonuclease activity creates a single-stranded nick and reverse transcriptase uses the nicked DNA to stimulate reverse transcription from the 3' end of the LINE RNA. Reverse transcription often fails to proceed to the 5' end, resulting in mostly truncated non-functional insertions. Thus, most LINE-derived repeats are short, with an average size of 900 bp for all LINE1 copies (IHGSC, 2001). c-mycER TAM All LINE repeats close to the integration site are truncated (maximum size = 170 bp) and have accumulated sequence mutations throughout evolution, and therefore are not active.

[0113] SINEs are short (100-400 bp) elements that harbor internal polymerase III promoters but do not code for proteins. The major SINE elements in this region are Alu, followed by MIR elements (inactive) and FLAM_C elements (fossil Alu monomers, inactive). More than 10% of the human genome consists of Alu repeats, which tend to self-associate in gene-rich regions (IHGSC, 2001). The only active SINEs are Alu elements. These do not code for any proteins and therefore rely on the L1 machinery of retrotransposition. Many SINEs are positioned to share the 3' end of LINE elements (Okada et al., 1997), presumably to aid or result from retrotransposition.

[0114] None of the SINE elements within the surveyed region are associated with the functionally active 3' end of the LINE repeat.

[0115] We conclude that no endogenous retrovirus is present in the vicinity of the integrated provirus, and thus activation of endogenous retrovirus as a result of integration seems an extremely unlikely outcome.

[0116] International Human Genome Sequencing Consortium (2001). Initial sequencing and analysis of the human genome. Nature 409: 860-921. Okada, N., Hamada, M., Ogiwara, I. & Ohshima, K. SINEs and LINEs share common 39 sequences: a review. Gene 205, 229±243 (1997).

[0117] 4. Neighboring genes The integration locus was analyzed to find other genes located in the vicinity of the integrated provirus. In addition to SPATA13, there are 10 known genes within the region of ±1 Mb of the integration site (see Table 2 and Figure 2). Analysis was performed for each of these.

[0118] 4.1 SGCG (distance=758,167bp) SGCG or gamma-sarcoglycan encodes the only transcript expressed in skeletal and cardiac muscle tissues. It is a component of the dystrophin glycoprotein complex (DGC), a group of proteins that span the sarcolemma and link actin to the extracellular matrix of muscle cells (Noguchi et al. 1995). Loss of SGCG causes a specific form of limb-girdle muscular dystrophy or sarcoglycanopathies, which are primarily associated with gastrocnemius muscle hypertrophy along with atrophy of the shoulder and surrounding muscles (Crosbie et al. 2000). Similarly, high expression of gamma-sarcoglycan in mice results in severe muscular dystrophy, with a significant loss of muscle mass and early lethality (Zhu et al. 2001).

[0119] Crosbie, RH; Lim, LE; Moore, SA; Hirano, M.; Hays , AP ; Maybaum , SW ; Collin , H. ; Device, SA; Stolle, CA; Fardeau , M. ; Tome, FMS; Campbell, K. P (2000) Molecular and genetic characterization of sarcospan: insights into sarcoglycan-sarcospan interactions. Hum. Malec. Genet. 9: 2019-2 Noguchi , S. ; McNally , EM ; Ben Othman, K.; Hagiwara , Y. ; Mizuno, Y.; Yoshida , M. ; Yamamoto , H. ; Bonnemann , CG ; Gussoni, E.; Denton, PH; Kyriakides , T. ; Middleton , L. ; Hentati, F.; Ben Hamida, M.; Nonaka, I.; Vance , JM ; Kunkel, LM; Ozawa. E.(1995) Mutations in the dystrophin-associated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy. Science 270: 819-821. Zhu X, Hadhazy M, Groh ME, Wheeler MT, Wellmann R, McNally EM. (2001) Overexpression of gamma-sarcoglycan induces severe muscular dystrophy. Implications for the regulation of Sarcoglycan assembly. J Biol Chem. 2001 Jun 15;276(24):21785-90. Epub 2001 Apr 3.

[0120] [Table 2]

[0121] 4.2 SACS(distance=649,642bp) The SACS (or SACSIN) gene encodes seven different transcripts by alternative splicing. Sacsin protein is highly expressed in the central nervous system, skeletal muscle, and at lower levels in the pancreas. The presence of a heat shock domain suggests a function for sacsin in chaperone-mediated protein folding (Engert et al. 2000). Deficiency of SACS is the cause of autosomal recessive Charlevoix-Saguenay spastic ataxia (ARSACS). ARSACS is an early-onset neurodegenerative disease with a high prevalence in the Charlevoix-Saguenay Lac-Saint-Jean region of Quebec. It is characterized by loss of sensory nerve conduction, slowing of motor nerve velocity, and hypermyelination of retinal nerve fibers.

[0122] Engert, JC; Berube, P.; Mercier, J.; Dore, C.; Lepage, P.; Ge, B.; Bouchard, J.-P.; Mathieu, J.; Melancon, SB; Schalling, M.; Lander, ES; an 11.5-kb ORF. Nature Genet. 24: 120-125, 2000.

[0123] 4.3 TNFRSF19(distance=407,228bp) TNFRSF19 encodes five distinct isoforms through alternative splicing. The protein encoded by this gene is a member of the TNF receptor superfamily. This receptor is highly expressed during embryonic development. It has been found to interact with TRAF family members and activate JNK signaling and NF-kB pathways when highly expressed in cells (Eby et al. 2000; Kojima et al. 2000). Although it lacks a death domain, this receptor is capable of programmed cell death through a caspase-independent mechanism (Wang et al. 2003). TNFRSF19 also plays a role in axon regeneration.

[0124] Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axon regeneration after injury. The Nogo receptor complex, consisting of Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75) and LING0-1, inhibits axon regeneration upon binding to such myelin components. The expression of p75 only on certain types of neurons and its temporal expression during development suggests that other receptors are involved in the NgR1 complex. TNFRSF19 is widely expressed in infant and adult neurons and can bind to NgR1 and displace p75 in the p75 / NgR1 / LING0-1 complex to activate the GTPase RhoA. This leads to stiffening of the actin cytoskeleton and growth cone collapse in the presence of myelin inhibitors (Park et al., 2005; Shao et al., 2005).

[0125] Eby MT, Jasmin A, Kumar A, Sharma K, Chaudhary PM. (2000) TAJ, a novel member of the tumor necrosis factor receptor family, activates the c-Jun N-terminal kinase pathway and mediates caspase-independent cell death. J Biol Chem. 2000 May 19;275(20):15336-42. Kojima, T., Morikawa, Y., Copeland, NG, Gilbert, DJ, Jenkins. NA, Senba, E. and Kitamura, T. (2000). TROY, a newly identified member of the tumor necrosis factor receptor superfamily, exhibits a homology with Edar and is expressed in embryonic skin and hair follicles. J. Biol. Chem. 275, 20742-20747 Park JB, Yiu G, Kaneko S, Wang J, Chang J, He XL, Garcia KC, He Z. (2005) A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron. 2005 Feb 3;45(3):345-51 Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N, Thill G, Levesque M, Sah D, McCoy JM, Murray B, Jung V, Pepinsky RB, Mi S. (2005) TAJ / TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron. 2005 Feb 3;45(3):353-9. Wang Y, Li X, Wang L, Ding P, Zhang Y, Han W, Dalong M. (2004) An alternative form of paraptosis-like cell death, triggered by TAJ / TROY and enhanced by PDCD5 overexpression J Cell Sci. 2004 Mar 15; 117(Pt 8): 1525-32.

[0126] 4.4 PCOTH (Distance = 186,070bp) PCOTH, or prostate collagen triple helix, encodes three distinct transcripts and putatively two distinct protein products. PCOTH expression is restricted to the testis and prostate, with a marked increase in expression in prostate cancer cells and their precursor, prostatic intraepithelial neoplasia (Ashida et al., 2004). High expression of PCOTH in healthy cells results in increased cell growth / division rates (Anazawa et al., 2005). Conversely, PCOTH siRNA in prostate tumor cells attenuated cell growth. PCOTH expression in prostate cancer cells was found to be associated with increased TAF-1β phosphorylation. TAF-1β (or SET), first identified as a fusion gene partner as a set-can gene in acute undifferentiated leukemia, was found to be a multitasking protein such as a potent inhibitor of protein phosphatase 2A, a target of granzyme A, an inhibitor of histone acetyltransferase, and a regulator of cell cycle transition, indicating that TAF-1β is a regulator of cell growth and proliferation (see refs 22-29 cited in Anazawa et al., 2005). PCOTH may somehow regulate the phosphorylation / activation of TAF-1β. TAF-1β is ubiquitously expressed in various tissues, whereas PCOTH expression is exclusively observed in testis, prostate, and prostate cancer. Thus, PCOTH may act as a prostate, testis, or prostate cancer specific regulator in controlling TAF-1β phosphorylation, and high expression of PCOTH may result in hyperphosphorylation of TAF-1β, which promotes cell survival.

[0127] Anazawa Y, Nakagawa H, Furihara M, Ashida S, Tamura K, Yoshioka H, Shuin T, Fujioka T, Katagiri T, Nakamura Y. (2005) PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-lbeta / SET. Cancer Res. Jun 1 ;65(11 ):4578-86. Ashida S, Nakagawa H, Katagiri T, et al. Molecular features of the transition from prostatic intraepithelial neoplasia (PIN) to prostate cancer: genome-wide geneexpression profiles of prostate cancers and PINs. Cancer Res 2004;64:5963-72.

[0128] 4.5 MIPEP (Distance = 193,884 BP) The MIPEP or mitochondrial intermediate peptidase gene encodes six different isoforms. The proteins carry out the final step of cleavage of certain classes of nuclear-encoded proteins that are targeted to the mitochondrial matrix or inner membrane (Lsaya et al 1991). Such proteins include (i) subunits of pyridine and flavin-binding dehydrogenases, (ii) iron-sulfur cluster-containing proteins and other nuclear-encoded subunits of respiratory chain complexes, (iii) proteins required for mitochondrial DNA replication and expression, and (iv) ferrochelatase, an enzyme that catalyzes iron binding in the final step of heme synthesis (Branda and Lsaya 1995, Chew 1997). This suggests that MIPEP is important for oxidative metabolism. MIPEP is also thought to have a role in mitochondrial iron homeostasis and possibly a regulatory effect on clinical severity in the neurodegenerative disease Friedreich's ataxia (Branda 1999).

[0129] Branda SS, lsaya G. (1995) Prediction and identification of new natural substrates of the yeast mitochondrial intermediate peptidase. J. Biol. Chem. 270 27366- 27373. Branda. SS, Yang Z, Chew A, lsaya G. (1999) Mitochondrial intermediate peptidase and the maintain yeast frataxin homolog together mitochondrial iron homeostasis in Saccharomyces cerevisiae. Hum. Molec. Genet. 8: I 099-1110. Chew A. Buck EA, Peretz S, Sirugo G. Rinaldo P, lsaya G. (l 997) Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP). Genomics 40: 493-496. lsaya G, Kalousek F, Fenton WA, Rosenberg LE. (1991) Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide. J. Cell Biol. 113 65-76.

[0130] 4.6 FLJ46358 (distance=129,175bp) The hypothetical protein FLJ46358 spans only 47 kb of the genome. No literature was found for the hypothetical protein FLJ46358. It was defined by seven cDNNEST clones (from pooled pancreas and spleen, astrocytoma cell lines, testis, and hippocampus). It does not contain any protein domains or unique Psort motifs. It is predicted by Psort to be localized in the cytoplasm. Sequence homology could not reveal any further information.

[0131] 4.7 PARP4(distance=337,591BP) Poly(ADP-ribose) polymerase 4 (PARP4) catalyzes the transfer of ADP-ribose moieties derived from NAO+ to various acceptor proteins, including PARP4 itself. This poly(ADP-ribosyl)ation of proteins is dramatically stimulated upon binding of the PARP4 DNA-binding domain (DBD) to single- or double-stranded breaks in DNA, and it is generally accepted that PARP plays an active role in the recovery of cells from DNA damage (Lindahl et al., 1995). Moreover, it has been described that:

[0132] PARP4 is quantitatively cleaved by caspase 3 during apoptosis induced by Fas ligand or DNA damage (Tewari et al., 1995; Nicholson et al., 1995). Inhibition of PARP4 results in genetic instability after DNA damage. Hela cells with constitutive high expression of dominant-negative PARP4 show reduced tumor formation in nude mice (Hans et al., 1999). This is most likely in accordance with increased tumor cell apoptosis in vivo. PARP4 is also present in the cytoplasm and is a component of the vault, a cytoplasmic ribonucleoprotein particle with a barrel-like twisted structure and two protruding caps (Zheng et al., 2005). The vault consists of at least six molecules of small non-coding RNA along with three proteins: major vault protein (MVP), PARP4, and two molecules of telomerase-associated protein 1 (TEP1). The function of the vault is unknown. However, they are certainly associated with chemotherapy resistance in primary tumors and various tumor cell lines and are generally recognized as negative prognostic factors for response to chemotherapy (Mossink et al., 2003). Although the detailed role of the vault in chemotherapy resistance is unclear, it has been suggested that the vault may alter the intracellular dynamics of anticancer drugs, for example, by sequestration of the drug in vesicles.

[0133] Lindahl T, Satoh MS, Poirier GG, Klungland A. (1995) Post-translational modification of poly(ADP-ribose) polymerase nduced by DNA strand breaks. Trends Biochem Sci. 1995 Oct;20(10):405-11. Mossink MH, van Zon A, Scheper RJ, Sonneveld P, Wiemer EA. (2003) Vaults: a ribonucleoprotein particle nvolved in drug resistance? Oncogene. 2003 Oct 20;22(47):7458-67. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, et al. (1995) Identification and inhibition of the ICE / CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37-43. Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM. (1995) Yama / CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995 Jun 2;81(5):801-9. Zheng CL, Sumizawa T, Che XF, Tsuyama S, Furukawa T, Haraguchi M, Gao H, Gotanda T, Jueng HC, Murata F, Akiyama S. (2005) Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes. Biochem Biophys Res Commun. 2005 Jan 7;326(1):100-7. Hans MA, Muller M, Meyer-Ficca M, Burkle A, Kupper JH. (1999) Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: evidence for increased tumor cell apoptosis in vivo. Oncogene. 1999 Nov 25;18(50):7010-5.

[0134] 4.8 ATP12A (distance=597,2248P) ATP12A encodes the alpha catalytic subunit of ouabain-sensitive H+ / K+ATPase, which catalyzes the hydrolysis of ATP associated with H(+) and K(+) ion exchange across the plasma membrane. It also mediates potassium absorption in various tissues. Tissue expression studies show significant levels in kidney and skin, intermediate levels in brain and placenta, and low levels in colon (Pestov et al., 1998).

[0135] Pestov NB, Romanova LG, Korneenko TV, Egorov MV, Kostina MB, Sverdlov VE, Askari A, Shakhparonov Ml, Modyanov NN. (1998) Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic alpha subunit. FEBS Lett. 1998 Dec 4;440(3):320-4.

[0136] 4.9 RNF17(distance=680,830bp) Ring finger 17 is specifically expressed in the testis (Wang et al. 2001) and is essential for spermatogenesis (Pan et al. 2001). It contains a Tudor domain and a RING finger motif. The Tudor domain is thought to function in RNA binding or protein interactions during RNA metabolism and / or transport (Ponting, 1997). The RING finger motif is present in many ubiquitin E3 ligases (Joazeiro and Weissman, 2000; Lorick et al., 1999). RNF17 interacts with four members of the Mad family (Mad1, Mxi1, Mad3, and Mad4), which are basic helix-loop-helix leucine zipper transcription factors that repress Myc-responsive genes by binding to the Max transcription factor (Yin et al., 1999). RNF17 has been shown to activate the transcription of Myc-responsive genes by sequestering Mad proteins (Yin et al., 2001;Yin et al., 1999). This redistribution of Mad proteins confers a "Mad null" phenotype and enhances the sensitivity of cells to several apoptotic stimuli, as well as high c-Myc expression.

[0137] Joazeiro, C. A. and Weissman. A. M. (2000). RI G finger proteins: mediators of ubiquitin ligase activity. Cell I 02, 549-552. Lorick. K. L.. Jensen. J.P., Fang. S.,Ong. A. M., Hatakeyama. S. and Weissman.. A. M. (1999). RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96. 11364-11369. Pan J, Goodheart M, Chuma S, Nakatsuji N, Page DC, Wang PJ. (2005) RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis. Development. 2005 Sep; 132(18):4029-39. Epub 2005 Aug 10. Ponting CP.(1997) Tudor domains in proteins that interact with RNA. Trends Biochem Sci. 1997 Feb;22(2):51-2. Wang, P. J., McCarrey, J. R .. Yang, F. and Page. D. C. (2001). An abundance of X-linked genes expressed in spermatogonia Xat. Genet. 27, 422-426. Yin, XY, Grove, LE and Prochownik, EV (2001). Mmip-2 / R.nf-17 enhances c-Myc function and regulates some target genes in common with glucocorticoid hormones. Oncogene 20. 2908-2917. Yin, X. Y. Gupta, K., Han, WP, Levitan, ES and Prochownik, EV (1999). Mmip-2. a novel RING finger protein that interacts with mad members of the Myc oncoprotein network. Oncogene 18. 6621-6634.

[0138] 4.10 CENPJ(distance=798,951bp) Centromeric protein J associates with the gamma-tubulin complex and was first identified thanks to its interaction with the cytoskeletal protein 4.IR-135 (Hung et al., 2000). Although CPAP appears to be a component of the centrosomal complex, the majority of CPAP is found in the soluble fraction, mainly in the cytoplasm and a small portion of the nucleus (Hung, 2000; Peng 2002). CPAP, which binds to STATS, translocates from the cytoplasm to the nucleus in response to prolactin-mediated activation of the JAK-STAT pathway and enhances STATS-dependent transcription (Peng 2002). CENPJ is also a coactivator of NF-kB that binds to the N-terminal region of RelA and may activate transcription through CBP (Koyanagi et al., 2005). NF-kB is a transcription factor important in various cellular phenomena such as inflammation, immune response, proliferation, and apoptosis.

[0139] Hung LY, Tang CJ, Tang TK. (2000) Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the gamma-tubulin complex. Mo / Cell Biol. 2000 Oct;20(20):7813-25 Koyanagi M, Hijikata M, Watashi K, Masui 0, Shimotohno K. (2005) Centrosomal P4.1-associated protein is a new member of transcriptional coactivators for nuclear factor-kappaB. J Biol Chem. 2005 Apr 1 ;280(13):12430-7. Epub 2005 Jan 31 Peng B, Sutherland KD, Sum EY, Olayioye M, Wittlin S. Tang TK, Lindeman GJ, Visvader JE. (2002) CPAP is a novel stat5-interacting cofactor that augments stat5-mediated transcriptional activity. Mol Endocrinol. 2002 Sep;16(9):2019-33.

[0140] 4.11 Conclusion Ten genes are within 1 Mb of the insertion site, with the closest gene, FLJ46358, at a distance of 129 Kb. It is highly unlikely that expression of genes at such a distance would be disrupted by retroviral insertion. None of the ten genes are known cancer genes, although two of them (PCOTH and PARP4) have been linked to cancer in the literature. Nevertheless, these two genes are 186 and 337 kb away from the insertion, respectively. It is highly unlikely and completely unprecedented that their expression would be disrupted by retroviral insertion at such a distance. Some of the other ten genes have been linked to disease in the literature, but there is no clear reason why they might pose a risk.

[0141] 5. Discussion The inserted provirus can induce oncogenic activation by a process of postinsertion mutagenesis. The question arises whether such activation could occur in CTX0E03 cells. The conclusion of the present analysis is that this possibility is extremely unlikely.

[0142] First, it is important to note the striking methodological differences between the generation of CTX0E03 cells and scenarios in which oncogenic activation occurred (e.g., the French X-SCID gene therapy trial). In such applications, the retroviral vector was itself the therapeutic agent, and a large number of blood progenitor cells were infected with the retrovirus. Each of these constitutes a separate insertion event. Thus, each patient was exposed to many potential mutagenic events. A selective process then follows, which spreads a subset of infected progenitors within the patient. Thus, at the start of the trial, there are many opportunities for unspecified genes to activate oncogenicity, revealing selective pressures that increase the tumorigenic potential. In contrast, the generation of CTX0E03 involves a single insertion event at a single identified locus, with no subsequent selection occurring. Thus, a priori, the probability of post-insertion mutagenesis is considerably reduced compared to gene therapy trials, and can be quantified in advance.

[0143] This evaluation of the oncogenic activation potential found the following: The CTX0E03 cell line has a retrovirus inserted into a gene with unknown oncogenic function. The nature of the insertion makes it unlikely to result in gene disruption or activation. The insertion does not fall within the vicinity of any endogenous retroviruses whose activation could induce significant oncogenic activation. There are no other genes other than the one being inserted that are close enough or have sufficient oncogenic potential to raise concerns about oncogenic activation.

[0144] Although the risk of oncogenic activation as a result of insertional mutagenesis can never be considered zero, since there are likely to remain oncogenic genes and mechanisms that remain to be discovered, the current state of knowledge makes it reasonable to conclude that the risk of oncogenic activation as a result of insertional mutagenesis in the CTX0E03 cell line is very low.

[0145] The use of the terms "a," "an," and "the," and similar terms in the context of the present description (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein, unless otherwise stated, is intended merely to facilitate a better understanding of the invention and does not pose a limitation on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0146] Embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of such embodiments may become apparent to those skilled in the art upon reading the foregoing specification. The inventors expect those skilled in the art to utilize such variations as appropriate, and the inventors intend the invention to be practiced other than as specifically described herein.

[0147] Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0148] All citations and reference documents are incorporated by reference in their entirety as if each individual disclosure was individually and explicitly incorporated. Examples of the invention of this application include the following: [1] A mammalian cell comprising a genetic modification in the SPATA13 gene on chromosome 13q12.12, (i) the cell is not a CTX0E03 cell; and / or (ii) the genetic modification is not an insertion of a cMYC-ER transgene; mammalian cells. [2] The cell described in [1] above, wherein the genetic modification is an insertion. [3] The cell described in [1] or [2] above, wherein the genetic modification is integration of an introduced gene. [4] The cell according to any one of [1] to [3] above, which is a human cell or an animal cell. [5] The cell according to any one of [1] to [4] above, which is a stem cell, a terminally differentiated cell, an immune cell, a T cell, a B cell or a Schwann cell. [6] The cell according to [5] above, which is a stem cell, a neural stem cell, a neural stem cell derived from fetal cortical tissue, a neural stem cell derived from a neural stem cell line, or a mesenchymal stem cell, or an iPS cell. [7] The cell of [6] above, when in accordance with [1](ii) above, which is a CTX0E03 cell genetically engineered by replacing the cMycER transgene with a different transgene. [8] a. Schwann cells with a cMycER transgene inserted within the SPATA13 gene on chromosome 13q12.12, or B T cells with a chimeric antigen receptor insertion within the SPATA13 gene on chromosome 13q12.12 The cell according to any one of the above [1] to [5], [9] The cell according to any one of [1] to [8] above, for use in therapy.

[10] A cell according to any one of [1] to [9] above, for use in a biotechnological process, optionally the process being the production of stem cells, proteins or microparticles.

[11] A pharmaceutical composition comprising the cell according to any one of [1] to [9] above.

[12] A method for generating a transgenic cell containing a stably integrated transgene, comprising the step of integrating the transgene at a site within the SPATA13 gene on chromosome 13q12.12.

[13] The method described in

[12] above, comprising CRISPR or TALEN gene editing technology.

[14] The method according to

[13] above, wherein the cell is a cell according to any one of [2] to

[10] above.

[15] The method according to any one of

[12] to

[14] above, wherein the insertion site is targeted using a gene therapy construct that is capable of and adapted to induce insertion of an exogenous introduced gene into the insertion site after administration to a human or animal.

[16] A cell obtained or obtainable by the method according to any one of

[12] to

[15] above.

[17] A nucleic acid molecule capable of inducing and adapted to induce insertion of a transgene into a site within the SPATA13 gene on chromosome 13q12.12 after administration to a human or animal.

[18] A cell according to [1] or

[18] above, or a nucleic acid according to

[17] above, for use in therapy.

[19] The cell or nucleic acid for use according to

[18] above, wherein the treatment is treatment of a disease or a genetic condition.

[20] The cell or nucleic acid for use according to

[19] above, wherein the treatment is treatment of cancer and optionally the cell is a T cell having a chimeric antigen receptor inserted within the SPATA13 gene on chromosome 13q12.12.

[21] The insertion site: a. Within an intron of the SPATA13 gene, b. Within the third intron of the SPATA13 gene, c. Within the third intron of the cDNA clone having Genbank accession number BX648244, D. Any nucleotide between 24,083,250 and 400 bp from the P terminus on chromosome 13q12.12; e. Any nucleotide between 24,083,300 and 350 bp from the P terminus on chromosome 13q12.12 f. Any of the nucleotides between 24,083,325 and 335 bp from the P terminus on chromosome 13q12.12; or g. Between nucleotides -24,083,331 and 24,083,332 bp from the P terminus on chromosome 13q12.12 The cell, composition, method, or nucleic acid molecule of any one of the preceding items,

Claims

1. 1. A transgenic human cell that is not an embryonic stem cell, comprising a transgene that has been inserted and integrated in vitro into the SPATA13 gene on chromosome 13q12.12, (i) the cell is not a CTX0E03 cell; and / or (ii) the inserted integrated transgene is not a cMYC-ER transgene; Transgenic human cells.

2. 2. The transgenic human cell of claim 1 which is a stem cell, a terminally differentiated cell, an immune cell, a T cell, a B cell or a Schwann cell.

3. 3. The transgenic human cell of claim 2, which is a stem cell, a neural stem cell, a neural stem cell derived from fetal cortical tissue, a neural stem cell derived from a neural stem cell line, or a mesenchymal stem cell, or an iPS cell.

4. The transgenic human cell of claim 3, which is a CTX0E03 cell genetically engineered by replacing the cMycER transgene with a different transgene when (ii) is followed.

5. a. Schwann cells with a cMycER transgene inserted within the SPATA13 gene on chromosome 13q12.12, or b. T cells with a chimeric antigen receptor inserted into the SPATA13 gene on chromosome 13q12.12 3. The transgenic human cell according to claim 1 or 2, 6. A transgenic human cell according to any one of claims 1 to 5, cultured ex vivo.

7. A cell culture comprising a transgenic human cell according to any one of claims 1 to 6 for use in a biotechnological process.

8. The cell culture of claim 7 , wherein the process is the production of stem cells, proteins or microparticles.

9. A pharmaceutical composition comprising the cell according to any one of claims 1 to 6.

10. 10. The pharmaceutical composition of claim 9, which, after administration to a human, provides an insertion site for the transgene within the human SPATA13 gene on chromosome 13q12.

12.

11. 10. A pharmaceutical composition according to claim 9 for use in therapy.

12. 12. The pharmaceutical composition of claim 11, wherein the treatment is treatment of a disease or genetic condition.

13. The pharmaceutical composition of claim 12, wherein the treatment is treatment of cancer.

14. The pharmaceutical composition of claim 12, wherein the cell is a T cell in which a chimeric antigen receptor has been inserted into the SPATA13 gene on chromosome 13q12.

12.

15. The insertion site is a. Within an intron of the SPATA13 gene, b. Within the third intron of the SPATA13 gene; c. Within the third intron of the cDNA clone having Genbank accession number BX648244; d. Any of the nucleotides between 24,083,250 and 400 bp from the P terminus on chromosome 13q12.12; e. Any of the nucleotides between 24,083,300 and 350 bp from the P terminus on chromosome 13q12.12; f. Any nucleotide between 24,083,325 and 335 bp from the P terminus on chromosome 13q12.12; or g. Between nucleotides -24,083,331 and 24,083,332 bp from the P terminus on chromosome 13q12.12 The pharmaceutical composition according to claim 10,

16. a. integrating a transgene into a site in the SPATA13 gene on chromosome 13q12.12; and / or b. The insertion site is targeted using a gene therapy construct that is inducible and adapted to induce insertion of an exogenous transgene into the insertion site using CRISPR or TALEN gene editing technology.

7. An in vitro method of generating a transgenic human cell according to any one of claims 1 to 6, comprising a stably integrated transgene comprising:

17. The insertion site is a. Within an intron of the SPATA13 gene, b. Within the third intron of the SPATA13 gene; c. Within the third intron of the cDNA clone having Genbank accession number BX648244; d. Any of the nucleotides between 24,083,250 and 400 bp from the P terminus on chromosome 13q12.12; e. Any of the nucleotides between 24,083,300 and 350 bp from the P terminus on chromosome 13q12.12; f. Any nucleotide between 24,083,325 and 335 bp from the P terminus on chromosome 13q12.12; or g. Between nucleotides -24,083,331 and 24,083,332 bp from the P terminus on chromosome 13q12.12 17. The method of claim 16, wherein:

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