Intronic targeted insertion of transgenes

Abstract

The invention relates to cells comprising a shutdown sequence and one or more transgene sequences inserted into both alleles of an intron of an endogenous gene. The invention relates to an optimised process for manufacturing of cells comprising a gene shutdown and, potentially, one or more transgene expression. The invention simplifies the shutdown process and improves shutdown efficiency.

Description

[0001] DESCRIPTION

[0002] TITLE: INTRONIC TARGETED INSERTION OF TRANSGENES

[0003] INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

[0004] SEQUENCE LISTING

[0005] The present application is filed with a Sequence Listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference.

[0006] BACKGROUND

[0007] Cellular therapy faces several challenges, among which safety and efficacy. Cell therapy products are meant to be transplanted in a host patient organism while originating from another individual, i.e. allotransplantation. To limit the phenomenon of allorejection, whereby transplanted cells are recognized as non-self and destructed by the immune system of the host patient, a medical research approach focuses on engineering cells prior to transplantation such that they become hypoimmunogenic. The engineering of hypoimmunogenic cells, often performed on pluripotent stem cells, requires the knockout of HLA-I and -II and overexpression of NK cell-inhibiting transgenes. This approach faces several challenges.

[0008] A challenge is the complexity of the genetic engineering approach due to the multiplicity of required genetic modifications and associated selection steps.

[0009] Another challenge relates to the enrichment in cells comprising the desired genetic modifications. For engineering of hypoimmunogenic cells, genetic modifications are performed using targeted insertion technology based on homologous recombination, using for example RNA-guided nucleases like MAD7. The nuclease introduces a specific DNA double strand break at a desired genomic target site thereby stimulating endogenous DNA repair pathways like homology-directed repair (HDR). If a DNA-based donor molecule (e.g., plasmid) containing a transgene flanked by sequences, which are homologous to the target site, is provided, it will serve as a template for homology-directed repair and the transgene will be inserted at the target site. Since HDR is a low frequent event in human somatic cells, enrichment is a critical step in any HDR-based genome editing process.

[0010] Another challenge is labour intensive efforts to identify cells with biallelic integration of genes of interest.

[0011] Further challenges include long and costly manufacturing of hypoimmunogenic cell therapy products. There is a need to further improve the manufacturing of gene edited cells for use in cellular therapy.

[0012] SUMMARY

[0013] The present invention relates to an exogenous sequence and to a genetic construct comprising the same. The present invention also relates to a cell comprising an exogenous sequence, to a cell population thereof, and to the use thereof in cellular therapy. The invention further relates to the manufacturing process and enrichment process of a cell population, said processes involving a genetic construct or an exogenous sequence per the invention.

[0014] The present invention brings several benefits that will be apparent from the present disclosure.

[0015] In one aspect, the present invention significantly simplifies and improves the manufacturing of a population of cells comprising a genetic modification of interest, such as a gene knock-in and a gene shutdown.

[0016] Also or alternatively, in a further aspect, the present invention is useful in the manufacturing of genetically engineered cells for use in cellular therapy, such as cell replacement therapy.

[0017] Also or alternatively, in a further aspect, the present invention is useful in the manufacturing of genetically engineered cells for use in gene therapy.

[0018] Also or alternatively, in a further aspect, the invention significantly reduces the number of successive gene editing steps.

[0019] Also or alternatively, in a further aspect, the invention significantly reduces the number of successive selection steps.

[0020] Also or alternatively, in a further aspect, the construct and / or method of the invention allow for direct selection of cellular clones that carry a bi-allelic targeted insertion of a transgene of interest, thereby avoiding clones with mono-allelic targeted insertion of a transgene of interest and / or mono-allelic shutdown of targeted endogenous gene of interest.

[0021] Also or alternatively, in a further aspect, the invention significantly improves the genetic engineering of mammalian cells which cannot be cloned, such as HSCs (hematopoietic stem cells) and T cells.

[0022] In an aspect, the invention relates to an exogenous sequence.

[0023] In an aspect, the invention relates to a genetic construct comprising said exogenous sequence. In an aspect, the invention relates to a cell comprising an exogenous inserted in an intron of an endogenous gene of interest and in both alleles thereof.

[0024] In an aspect, the invention relates to a cell population.

[0025] In an aspect, the invention relates to a pharmaceutical product.

[0026] In an aspect, the invention relates to medical uses of said call, cell population and / or pharmaceutical product.

[0027] In an aspect, the invention relates to the manufacture of cells or a cell population thereof, comprising a gene editing step wherein said cell(s) is(are) subjected to the targeted insertion of said genetic construct into an intronic locus of an endogenous gene thereof.

[0028] In an aspect, said exogenous sequence comprises a shutdown sequence and, optionally, one or more transgene sequence(s). In an aspect, said shutdown sequence comprises a splice acceptor sequence, a stop codon and a polyA sequence, and may further comprise a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon. In an aspect, said transgene sequence(s) comprises a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0029] Further benefits brought by the present invention include:

[0030] Also or alternatively, in another aspect, the use of the construct in an enrichment process allows for the selection of cells (and enrichment) comprising both the biallelic knock- in (KI) and the biallelic shutdown in one same step.

[0031] Also or alternatively, in another aspect, the invention significantly improves the success of gene editing strategies.

[0032] Also or alternatively, in a further aspect, the invention provides a much improved success rate for biallelic insertion.

[0033] Also or alternatively, in a further aspect, the invention allows to achieve gene shutdown and transgene knock-in at the same time, i.e. in same step.

[0034] Also or alternatively, in a further aspect, the invention allows for easy sorting of cells carrying the desired knock-in and shutdown. Indeed, as per the invention, biallelic knock-in is the only way to achieve the desired gene shutdown, therefore cells with gene shutdown necessarily carry a biallelic knock-in as well.

[0035] Also or alternatively, in a further aspect, the invention simplifies the enrichment in cells comprising a genetic modification of interest. The invention allows for direct enrichment of cells with bi-allelic targeted insertion, without enriching for cells carrying mono-allelic transgene insertion mono-allelic shutdown of a target gene by NHEJ (non homogolous end joining) and / or random integration events.

[0036] Also or alternatively, in a further aspect, the invention allows for stable expression of knocked-in transgene, as biallelic knock-in allows for more stable expression than monoallelic transgene knock-in.

[0037] Also or alternatively, in a further aspect, the invention allows for improved expression of the knocked-in gene, as biallelic knock-in of a gene allows for more expression than monoallelic knock-in of the same.

[0038] Also or alternatively, in a further aspect, the invention improves the manufacturing of genetically engineered cells of use in the field of cell therapy, such as cell replacement therapy, in particular where said genetic engineering encompasses both gene shutdown, i.e. endogenous gene disruption, and transgene knock-in, i.e. exogenous gene insertion.

[0039] Also or alternatively, in a further aspect, the invention improves the manufacturing of genetically engineered cells in the field of CAR-T cell therapy. The construct and / or method of the invention can also be applied to cells that cannot be cloned ex vivo, such as multipotent stem cells (e.g. hematopoietic stem cells) and differentiated hematopoietic cells (T cells, macrophages, NK cells) used in CAR-T cell therapy, thereby enabling a direct enrichment of bi-allelic edited cells and increasing homogeneity of any cell product (CAR T cells, CAR Tregs, CAR macrophages, CAR NK cells). In the context of CAR-T cell therapy, blood cells such as T cells may be retrieved from a patient, genetically engineered ex-vivo to disrupt endogenous T cell receptor (TCR) gene(s), and transplanted back to the same patient (autologous transplantation), and cells usually cannot be cloned. The invention improves the enrichment in cells carrying the desired genetic edits prior to transplantation.

[0040] Also or alternatively, in a further aspect of the invention, on-target cleavage without targeted insertion will not generate any mutant forms of the encoded protein.

[0041] The invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments and the overall content of the present patent document.

[0042] BRIEF DESCRIPTION OF DRAWINGS

[0043] Fig. 1 shows a graphical representation of gene editing with various constructs. Fig. 1 is further explained in the specification. Fig .2 shows a graphical representation of an example of exogenous sequence comprising two transgene coding sequences, inserted in a cell genome. Fig.2 is further explained in the specification.

[0044] Fig.3 shows a graphical representation of the targeted insertion, in an intron of an endogenous gene, of a genetic construct comprising an exogenous sequence comprising a shutdown sequence, and of the potential outcomes thereof at the transcription and translation level. Fig.3 is further explained in the specification.

[0045] Fig.4A and B show graphical representations of targeted insertion, in an intron of an endogenous gene, of a genetic construct comprising an exogenous sequence comprising a shutdown sequence and a transgene sequence, and of the potential outcomes thereof at the transcription and translation level. Fig.4 is further explained in the specification.

[0046] Fig. 5 A to J show FACS results on cells upon targeted insertion of genetic constructs comprising various exogenous sequences listed in Table 5. Fig.5 is further explained in Example 1 .

[0047] Fig. 6 shows a graphical representation of the targeted insertion, in intron 1 of endogenous B2M gene, of a genetic construct comprising an exogenous sequence comprising a shutdown sequence and two transgene sequences.

[0048] Fig. 7A and B show FACS results on beta-like cells derived from a stem cell which has been subjected to the targeted insertion of genetic a construct comprising exogenous sequence #1 1 as listed in Table 5. Fig.7 is further explained in Example 3.

[0049] Fig. 8 shows FACS results on beta-like cells derived from a stem cell which has been subjected to the targeted insertion of genetic a construct comprising exogenous sequence #4 as listed in Table 5. Fig.8 is further explained in Example 3.

[0050] Fig. 9 shows FACS results on beta-like cells derived from a stem cell which has been subjected to the targeted insertion of a genetic construct comprising exogenous sequence #1 1 as listed in Table 5. Fig.9 is further explained in Example 4.

[0051] Fig. 10 shows TCR dextramer assay results on beta-like cells derived from a stem cell which has been subjected to the targeted insertion of genetic a construct comprising exogenous sequence #1 1 as listed in Table 5. Fig.10 is further explained in Example 4.

[0052] Fig. 11 A-B schematically illustrates exon editing of a cell surface protein using Non Homologous End Joining (NHEJ) (Fig .11 .A) and FACS sorting thereof (Fig .11 B) after gene editing. Fig. 12 schematically illustrates exon editing of cell surface protein and transgene insertion using Homology-Directed Repair (HDR) (Fig.12.A) and FACS sorting thereof (Fig.12 B) after gene editing.

[0053] Fig. 13 schematically illustrates intron editing of cell surface protein using NHEJ (Fig.13. A) and FACS sorting thereof (Fig.13 B) after gene editing.

[0054] Fig. 14 schematically illustrates intron editing of cell surface protein and transgene insertion using HDR (Fig.14.A) and FACS sorting thereof (Fig.14 B) after gene editing.

[0055] Fig.15 shows the proportion of successful integration, i.e. of biallelic targeted intronic integration.

[0056] DESCRIPTION

[0057] In an aspect, the invention relates to a sequence, also called exogenous sequence.

[0058] In an aspect, the invention relates to a cell comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof.

[0059] In an aspect, the invention relates to a cell comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof and obtained by a method of manufacturing as defined herein.

[0060] In an aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof.

[0061] In an aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said cell population has been obtained by a method of manufacturing as defined herein.

[0062] In an aspect, the invention relates to a method of manufacturing a cell or a cell population as defined herein.

[0063] The expression “exogenous sequence” refers to a nucleotide sequence that is not naturally present in a given location in a cell’s genome.

[0064] The expression “endogenous gene” refers to a nucleotide sequence that is naturally present in a given location in a cell’s genome and which can express a protein upon effect of cellular transcription and translation machineries. In a preferred embodiment, the endogenous gene of interest comprises at least one intron and two exons. The terms “intron”, “exon” and “allele” mean as generally defined in the fields of genetic and biotechnology.

[0065] Exogenous sequence:

[0066] In an aspect, the exogenous sequence comprises

[0067] • a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0068] In an embodiment, the exogenous sequence comprises

[0069] • a splice acceptor (SA) sequence, a stop codon such as a Tristop sequences and preferably two or more stop codons such as two successive Tristop (triple stop) sequences, a coding sequence and two or more successive polyA sequences, such as three or more, and preferably four or more polyA sequences, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequence(s).

[0070] In an embodiment, the exogenous sequence comprises

[0071] • a splice acceptor (SA) sequence, a coding sequence, a stop codon such as a Tristop sequences and preferably two or more stop codons such as two successive Tristop (triple stop) sequences, and two or more successive polyA sequences, such as three or more, and preferably four or more polyA sequences, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and stop codon and before said polyA sequence(s).

[0072] In an embodiment, the exogenous sequence comprises a splice acceptor (SA) sequence, two successive triple stop sequences, a coding sequence which comprises exon 3 of B2M gene or a portion thereof, four successive polyA sequences, such as polyA sequence derived from SV40, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequences, such as the sequence SA-Tristopx2- B2MExon3-sv40pA(Short)x4.

[0073] In an embodiment, the exogenous sequence comprises a splice acceptor (SA) sequence, a coding sequence which comprises exon 3 of B2M gene or a portion thereof, two successive triple stop sequences, four successive polyA sequences, such as polyA sequence derived from SV40, and optionally a 3’UTR sequence or a portion thereof located after said triple stop sequences and before said polyA sequences, such as the sequence SA- B2MExon3- T ristopx2-sv40pA(Short)x4.

[0074] In another aspect, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0075] In another aspect, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0076] In an alternative embodiment, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence located after said splice acceptor sequence and before said stop codon and a 3’UTR sequence or a portion thereof located after said stop codon and before said polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0077] In another aspect, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence, a stop codon, a 3’UTR sequence or a portion thereof and a polyA sequence, and II. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0078] Shutdown sequence:

[0079] The first sequence in the exogenous sequence may be called a “shutdown sequence”.

[0080] Upon insertion of the exogenous sequence in the intron of an endogenous gene in a cell, an effect of the first sequence is to prevent protein expression from said endogenous gene.

[0081] In the first sequence, the so-called “coding sequence” prevents the expression of a functional protein upon transcription-translation together with the preceding exon(s) from the endogenous gene of interest, it interrupts the amino acid sequence of the endogenous protein. After transcription into mRNA, said “coding sequence” is translated into one or more amino acids in continuation of the amino acid sequence in progress (from the translation of said preceding endogenous exon(s)). This results in a truncated hence non-functional protein which gets degraded in the cell. In essence, the “coding sequence” in the first sequence ensures together with the stop codon that the translation of the endogenous gene is aborted, thereby shutting down its expression.

[0082] Transgene sequence:

[0083] The second and potential further sequences in the exogenous sequence may be called “transgene sequences".

[0084] Upon insertion of the exogenous sequence in the intron of an endogenous gene in a cell, the transgene sequence(s) has(have) the effect to allow the expression of a protein (proteins) of interest.

[0085] Further, said exogenous sequence, first and second sequences are nucleotide sequences. They may be synthetic sequences, i.e. artificially constructed prior to their insertion in a cell.

[0086] The shutdown sequence (i) is located in 5’ position of the transgene sequence(s). The transgene sequence(s) may come in any order relative to each other.

[0087] In an embodiment, sequence (ii) may be placed either in 5’ or in 3’ of sequence (iii).

[0088] In an embodiment, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence comprising exon 3 or a portion thereof of human B2M gene, such as a portion of sequence SEQ ID NO 4 , a stop codon, a 3’UTR sequence or a portion thereof and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence comprising a B2M-HLA fusion gene, such as B2M-HLA-E fusion gene and / or B2M- HLA-G fusion gene, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, wherein sequence (i) is placed 5’ of sequences (ii) and (iii), and wherein sequence (ii) may be placed in 5’ or 3’ position of sequence (iii).

[0089] Splice acceptor (SA):

[0090] The term “splice acceptor" has the same meaning as generally acknowledged in the fields of genetic and biotechnology. The primary function of a splice acceptor is to remodel the exons combination in the mRNA (messenger RNA) comprising it, as post-translation maturation thereof, by merging with a splice donor sequence elsewhere on the same mRNA.

[0091] The splice acceptor (SA) may be any splice acceptor sequence, such as engineered ones or natural ones. Exemplary splice acceptor sequences are known to those of skill in the art and include, by way of example, sequence SEQ ID NO 01 , sequence SEQ ID NO 02, sequence SEQ ID NO 03, or a sequence AG. In an embodiment, the splice acceptor sequence is AG. In an embodiment, the splice acceptor sequence is SEQ ID NO 01 . (Table 1)

[0092] Coding sequence in the first sequence (i):

[0093] The coding sequence comprised in the first sequence does not allow expression of a functional protein upon integration and expression together with the preceding exon(s) from the endogenous gene of interest.

[0094] In an embodiment, the coding sequence in the first sequence comprises between 1 to 50 nucleotides, preferably 1 to 40, 1 to 30, 1 to 20, 5 to 20 nucleotides.

[0095] In an embodiment, the coding sequence in the first sequence is or comprises human B2M gene exon 3 or a portion thereof , such as SEQ ID NO 4. (Table 1)

[0096] In an embodiment, a portion of a coding sequence in the first sequence comprises between 1 to 50 nucleotides, such as 1 to 40, 1 to 30, 1 to 20, 6 to 20, 1 to 12, 1 to 9 or 1 to 6 nucleotides.

[0097] In an embodiment, the coding sequence in the first sequence (i) improves the shutdown of the targeted endigenous gene. 3’UTR sequence:

[0098] In the present invention, the exogenous sequence may comprise a 3’UTR sequence or a portion of a 3’UTR sequence. The term “3’UTR (3' untranslated region)” has the same meaning as generally acknowledge in the fields of genetic and biotechnology. The 3' untranslated region (3'UTR) of an mRNA is a region that follows the coding sequence and precedes the poly(A) tail. It plays a critical role in the post-transcriptional regulation of gene expression. It is involved in regulating mRNA stability, translation efficiency, and localization. This region can contain various regulatory elements, such as binding sites for RNA-binding proteins and microRNAs (miRNAs), which influence the fate of the mRNA molecule. A “3’UTR sequence” typically comprises a mRNA terminator sequence and may further comprise some regulatory elements (Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell (4th ed.); Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Bretscher, A., Ploegh, H., Amon, A., & Martin, K. C. (2016). Molecular Cell Biology (8th ed.).). Exemplary 3’UTR sequences are known to those of skill in the art. By ay of example, the sequence of TP53 3’UTR is provided as sequence SEQ ID NO 05 (Table 1).

[0099] Stop codon:

[0100] The term “stop codon” has the same meaning as generally acknowledged in the fields of genetic and biotechnology. A stop codon is a triplet of nucleotides in mRNA that signals the end of translation. The three stop codons - UAA, UAG, and UGA - do not correspond to any amino acids and thus cause the ribosome to release the newly synthesized protein (Brown, T. A. (2010). Gene Cloning and DNA Analysis: An Introduction (6th ed.)). The primary function of a stop codon is to act as a translational stop signal to the translation machinery (such as ribosomes).

[0101] The stop codon may be any stop codon sequence known in the art.

[0102] In an embodiment, the stop codon sequence is selected from TAA, TAG, TGA nucleotide sequences, which correspond to UAA, UAG, UGA ribonucleotide sequences.

[0103] In another embodiment, the stop codon sequence comprises more than one stop codon sequence, such as in different reading frames. In an embodiment, the stop codon sequence comprises one or more triple frame stop sequence(s), which may be abbreviated as “tristop” or “TFS”.

[0104] In an embodiment, the stop code comprises 2 triple frame stop sequences, such a stop codon may comprise a sequence SEQ ID NO 06. (Table 1). The stop codon of the first sequence, of the second sequence and of any potential further sequence are selected independently of each other, such that they may be the same or different stop codon sequences.

[0105] PolyA sequence

[0106] The term “polyA sequence”, abbreviated “pA”, has the same meaning as generally acknowledged in the fields of genetic and biotechnology. The polyA sequence is a stretch of adenine nucleotides, typically 50-250 bases long, added to the 3' end of a pre-mRNA molecule during RNA processing in eukaryotic cells. This modification occurs in the nucleus and is catalysed by the enzyme poly(A) polymerase. The primary function of a polyA sequence is to act as a transcriptional stop signal to the transcription machinery (such as RNA polymerase). It also plays several other critical roles, including enhancing the stability of the mRNA, facilitating its export from the nucleus to the cytoplasm, and aiding in the initiation of translation. The presence of the polyA sequence is a hallmark of mature mRNA and is essential for efficient gene expression (Lewin, B. (2008). Genes IX.; Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Bretscher, A., Ploegh, H., Amon, A., & Martin, K. C. (2016).

[0107] Molecular Cell Biology (8th ed.)).

[0108] The polyA sequence may be or comprise any well-known polyA sequence. In an embodiment, the polyA sequence comprises one or more sequences selected from SV40 polyA (from Simian Virus 40) comprising SEQ ID NO 07, bGH polyA (from bovine Growth Hormone) comprising SEQ ID NO 08, ‘‘polyA short sequence” such as sequence SEQ ID NO 09, ‘‘polyA synthetic sequence” such as sequence SEQ ID NO 10 and / or sequences derived therefrom and combinations thereof. (Table 1)

[0109] In an embodiment, the polyA sequence comprises more than one polyA sequence, such as 2, 3 or 4 polyA sequences.

[0110] In an embodiment, the polyA sequence is or comprises 2 SV40 polyA sequences. In an embodiment, the polyA sequence is or comprises 4 SV40 polyA sequences.

[0111] In an embodiment, the polyA sequence is or comprises 2 bGH polyA sequences. In an embodiment, the polyA sequence is or comprises 4 bGH polyA sequences.

[0112] The polyA sequences of the first sequence, of the second sequence and of any potential further sequence are selected independently of each other, such that they may be the same or different polyA sequences.

[0113] Promoter: The term “promoter” has the same meaning as generally acknowledged in the fields of genetic and biotechnology. A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, upstream on the DNA. In mammalian cells, promoters contain specific DNA sequences that are recognized by transcription factors and RNA polymerase II, which are necessary for the initiation of transcription ((Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell (4th ed.J; Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Bretscher, A., Ploegh, H., Amon, A., & Martin, K. C. (2016). Molecular Cell Biology (8th ed.)). The promoter may be any promoter known in the art.

[0114] In a preferred embodiment, the promoter is one known as a “strong promoter”, i.e. allowing high level of gene expression and / or ubiquitous and / or constant gene expression, and may be selected from a CAG (CMV early enhancer chicken beta actin) promoter, a UBC (Ubiquitin C) promoter and EF1-alpha (human elongation factor-1 alpha) promoter.

[0115] In an embodiment, the promoter is or comprises the CAG promoter sequence SEQ ID NO 11 . In an embodiment, the promoter is or comprises the UBC promoter sequence SEQ ID NO 12. In an embodiment, the promoter is or comprises the EF1-alpha promoter sequence SEQ ID NO 13. (Table 1)

[0116] The promoter of the first sequence, of the second sequence and of any potential further sequence are selected independently of each other, such that they may be the same or different promoters.

[0117] Table 1 : Non limiting examples of nucleotide sequences as splice acceptor, coding sequence, stop codon, polyA or promoter

[0118]

[0119] Transgene coding sequence:

[0120] In a preferred embodiment, the transgene coding sequence(s) comprised in the second sequence (ii) and in potential further sequences (iii) of the exogenous sequence allows expression of a functional protein upon integration of the exogenous sequence in an intron of the endogenous gene of interest.

[0121] The transgene coding sequence may encode any protein(s) of interest. The skilled person is able to select the transgene coding sequence(s) of interest based on a desired cell characteristic.

[0122] In an embodiment, especially for use in CAR-T (chimeric antigen receptor) cell therapy, the transgene coding sequence may be one encoding a TCR (T-cell receptor).

[0123] In an embodiment, the biallelic insertion of said transgene coding sequence of said second sequence (ii) and of said potential further sequences (ill) improves the shutdown of said endogenous gene.

[0124] In an embodiment, of particular interest in the field of cellular therapy, said transgene coding sequence(s) is selected from the list consisting of

[0125] CD55 gene, encoding CD55 protein (Cluster of Differentiation 55 also known as Complement decay-accelerating factor or DAF) wild type or an analogue thereof, PDL1 gene, encoding PDL1 protein (Programmed death-ligand 1) wild type or an analogue thereof,

[0126] CD47 gene, encoding CD47 protein (Cluster of Differentiation 47 also known as integrin-associated protein IAP) wild type or an analogue thereof,

[0127] HLA-E gene, encoding HLA-E (human leucocyte antigen-E) protein, wild type or an analogue thereof,

[0128] HLA-F gene, encoding HLA-F (human leucocyte antigen-F) protein wild type or an analogue thereof; a B2M-HLA fusion gene, encoding a B2M-HLA fusion protein, such as a B2M-HLA-E and / or a B2M-HLA-G fusion protein(s), such as B2M / HLA-E*0101 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein, optionally with a (G4S)4 linker and a signal peptide, or B2M / HLA-E*0103 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein, optionally with a (G4S)4 linker and a signal peptide; a suicide switch gene encoding a suicide switch protein, and combinations thereof. Above mentioned genes and proteins are defined in the art. Sequences are further provided in Table 2 herein.

[0129] Table 2: Non limiting examples of nucleotide sequences as transgene coding sequence: The sequence of human wild type CD55 is referenced as P08174-1 in Uniprot.

[0130] The sequence of human wild type PDL1 is referenced as NP_001134823.1 in NCBI.

[0131] As used herein, “B2M-HLA fusion protein”, or “B2M-HLA genetic fusion protein”, means a protein comprising a B2M (beta-2-microglobulin) protein and a HLA protein fused into one same protein. Further definitions and embodiments, including sequences, for “B2M-HLA”, “B2M-HLA-E” and “B2M-HLA-G" genes and proteins can be found in WO / 2022 / 129472 and WO / 2022 / 136215 and are incorporated herein by reference.

[0132] As used herein, the term “HLA” stands for human Leucocyte Antigen and refers to the well- known HLA system responsible for the regulation of the immune system in mammalians.

[0133] A suicide switch is a genetic construct or encoded protein(s) therefrom which allows selective destruction of cells comprising it. Suicide switches are used to improve the safety of cell therapy products, and particularly to prevent proliferation of cell therapy products in the body of a host patient.

[0134] In an embodiment, the transgene coding sequence encodes a marker protein, such as SEQ ID NO 16 encoding a GFP protein, or SEQ ID NO 17 encoding mCherry protein, or analogues thereof and combination thereof.

[0135] Table 3: Non limiting examples of nucleotide sequences as transgene coding sequence:

[0136] Further elements:

[0137] The exogenous sequence may comprise further elements. Said further sequences may be selected from any elements known in the field of genetic engineering and with various functions.

[0138] By way of example, the exogenous sequence may further comprise:

[0139] - a chromatine opening element such as UCOE (Ubiquitous chromatin opening element), such as sequence SEQ ID NO 18 (Table 4), or UCOE462 which is a shorter version of UCOE; - a ribosome skipping element, such as T2A element and / or F2A element, which may be used for example right after a promoter to allow ribosomes to skip from one reading frame to the next reading frame, such as sequence SEQ ID NO 19 and / or SEQ ID NO 20 (Table 4); - an insulator element, which may be used for example to add some distance in between 2 promoters and / or to secure or enhance transgene expression in presence of two transgenes, such as cHs4 insulator sequence for example comprising sequence SEQ ID NO 21 or such as Smar 4 buffer sequence for example comprising sequence SEQ ID NO 22 (Table 4); or - combination(s) thereof.

[0140] Table 4: Non limiting examples of nucleotide sequences as further elements:

[0141] In an embodiment, the exogenous sequence comprises one transgene coding sequence encoding a protein of interest.

[0142] In an embodiment, the exogenous sequence comprises: i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0143] By way of example, Fig. 1 illustration (C) shows an embodiment wherein the exogenous sequence comprises: i. a first sequence comprising a splice acceptor indicated as “SA”, a coding sequence indicated as “exon 3", a 3’UTR sequence or a portion thereof indicated as “3’UTR”, a stop codon indicated as “2x TFS” which comprises 2 triple frame stop sequences, a polyA sequence indicated as “4x polyA” which comprises 4 polyA sequences, and a chromatin opening element indicated as “UCOE”; and ii. a second sequence comprising a promoter indicated as “promotor”, a transgene coding sequence indicated as “transgene”, a stop codon not indicated and in this case included in the “transgene” sequence, and a polyA sequence indicated as “pA”.

[0144] In another embodiment, the exogenous sequence comprises more than one, such as two, three or four transgene coding sequences encoding proteins of interest, generally a different protein from each transgene.

[0145] In an embodiment, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequencea stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. a third and a fourth further sequence, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0146] In another embodiment, the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and iil. a third sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0147] By way of example, Fig. 1 illustration (D) shows an embodiment wherein the exogenous sequence comprises: i. a first sequence (the shutdown sequence) comprising a splice acceptor indicated as “SA”, a coding sequence indicated as “exon 3”, a 3’UTR sequence or a portion thereof indicated as “3’UTR”, a stop codon indicated as “2x TFS” which comprises two triple frame stop sequences, a polyA sequence indicated as “4x polyA” which comprises four polyA sequences, and a chromatin opening element indicated as “UCOE”; ii. a second sequence (a transgene sequence) comprising a promoter indicated as “promotori ”, a transgene coding sequence indicated as “transgenel”, a stop codon not indicated and in this case included in the “transgenel” sequence, a polyA sequence indicated as “pA”, and an insulator element indicated as “2x cHS4” which comprises two cHS4 insulator sequences; and iii. a third sequence (a transgene sequence) comprising a promoter indicated as “promotor 2”, a transgene coding sequence (indicated as “transgene 2”, a stop codon not indicated and in this case included in the “transgene 2” sequence, a polyA sequence indicated as “pA" and an insulator element indicated as “2x Smar 4” which comprises two Smar 4 sequences.

[0148] By way of example, Fig. 2 illustrates an example of exogenous sequences comprising two transgene coding sequences.

[0149] In the example named “Gene Edit #1”, the exogenous sequence comprises: i. a first sequence not shown comprising a splice acceptor, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon, and a polyA sequence; ii. a second sequence comprising a CAG promoter indicated as “GAG”, a transgene coding sequence indicated as “HLA-E(or G)” and encoding a HLA-E (or a HLA-G) protein, a stop codon not shown and in this case included in the 5’ end of the transgene coding sequence, a polyA sequence not shown, and possibly an insulator element not shown; and iii. a third sequence comprising a EF1a promoter indicated as “EF1a”, a transgene coding sequence indicated as “CD55" and encoding a CD55 protein, a stop codon not shown and in this case included in the 5’ end of the transgene coding sequence, a polyA sequence not shown, and possibly a further element not shown.

[0150] In an aspect, the invention relates to a cell comprising an exogenous sequence as defined herein.

[0151] In an aspect, the invention relates to a cell comprising an exogenous sequence as defined herein located within an intron of an endogenous gene of interest and in both alleles thereof. An effect of the presence of said exogenous sequence within an intron of an endogenous gene of interest in the cell is the shutdown of the expression of said endogenous gene.

[0152] An effect of the presence of said exogenous sequence within an intron of both alleles of said endogenous gene of interest in the cell is the complete shutdown of the expression of said endogenous gene in the cell. In other words, such biallelic insertion of the exogenous gene ensures absence of protein expression from said endogenous gene.

[0153] Endogenous gene:

[0154] The endogenous gene of interest depends on the desired cell phenotype and on the intended use of the cell. The endogenous gene of interest may be any gene.

[0155] In an embodiment, the endogenous gene of interest is any gene, such as any gene which is beneficial or neutral to be shutdown in a given context.

[0156] In a preferred embodiment, the endogenous gene of interest comprises at least one intron and two exons.

[0157] In an embodiment, the endogenous gene of interest encodes a cell surface protein.

[0158] In an embodiment, the endogenous gene of interest is not an essential gene. In other words, the endogenous gene of interest does not encode a protein which is essential to cell survival. In an embodiment, the endogenous gene of interest is not HPRT (Hypoxanthine Phosphoribosyltransferase 1) gene.

[0159] In an embodiment, especially for use in CAR-T cell therapy, the endogenous gene of interest may be the endogenous TCR gene.

[0160] In an embodiment, especially for use in cell replacement therapy, the endogenous gene of interest may be one that contributes to the immune rejection thereof.

[0161] In a further embodiment, the endogenous gene of interest may be selected from the list consisting of B2M (p2 microglobulin) gene, CIITA (class II, major histocompatibility complex transactivator) gene, TRAC (T-cell receptor alpha constant) gene, HLA-A gene, HLA-B gene, HLA-C gene, CD155 (also known as poliovarius receptor or PVR) gene, CD122 (also known as interleukine-2 receptor subunit beta or IL2RB) gene and B7-H3 (B3 homolog 3, also known as cluster of differentiation 276 or CD276) gene.

[0162] In a particular embodiment, the endogenous gene of interest is the B2M gene.

[0163] In a particular embodiment, the endogenous gene of interest is the CIITA gene.

[0164] In a particular embodiment, the endogenous gene of interest is the CKD1 gene. When the endogenous gene of interest comprises more than one intron, the exogenous sequence may be located within any one intron in said gene, such as the first, second, third or any further intron of said endogenous gene of interest. In a preferred embodiment, the exogenous sequence is located within the first intron in the endogenous gene of interest. In a preferred embodiment, the exogenous sequence is located within the second intron in the endogenous gene of interest. In a further embodiment, the exogenous sequence is located within the third intron in the endogenous gene of interest.

[0165] As used herein, the first intron of a given gene may be referred to as intron 1 and the second intron may be referred to as intron 2, etc..

[0166] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of B2M endogenous gene. In a preferred embodiment, it is located within intron 1 of B2M endogenous gene.

[0167] In another embodiment, said exogenous sequence is located within intron 1 or intron 2 of CIITA endogenous gene. In a preferred embodiment, it is located within intron 1 of CIITA endogenous gene.

[0168] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of TRAC endogenous gene.

[0169] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of HLA-A endogenous gene.

[0170] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of HLA-B endogenous gene.

[0171] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of HLA-C endogenous gene.

[0172] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of CD155 endogenous gene.

[0173] In an embodiment, said exogenous sequence is located within intron 1 or intron 2 of CD122 endogenous gene.

[0174] The cell may be any cell type.

[0175] In an aspect, the cell is a diploid cell, meaning that the cell comprises in its genome two copies of each autosome, hence comprises two copies, also called alleles, of each autosomal endogenous gene.

[0176] In an embodiment, the cell is a mammalian cell. In a preferred embodiment, the cell is a human cell. In an embodiment, the cell is a genetically engineered cell, such as a genetically engineered human cell.

[0177] As used herein, the term “genetically engineered cell” refers to a cell, the genome of which has been artificially modified usually using laboratory based technologies.

[0178] In an aspect, the invention relates to a cell comprising an exogenous sequence as defined herein, wherein said exogenous sequence is located within an intron of an endogenous gene of interest in the genome of said cell and in both alleles thereof, and wherein said cell has been obtained by the targeted insertion of said exogenous sequence within said intron.

[0179] In an aspect, the invention relates a method of manufacturing a cell as defined herein, wherein said method comprises the targeted insertion of an exogenous sequence as defined herein within an intron of an endogenous gene of interest in the genome of said cell.

[0180] In an embodiment, said targeted insertion has been obtained by homologous recombination between (i) a genetic construct comprising said exogenous sequence flanked by homology arms to a target insert site and (ii) said target insertion site located within said endogenous gene of interest in the genome of said cell.

[0181] In an embodiment, said homologous recombination has further involved a gene editing tool such as TALEN or a CRISPR nuclease, such as Cas9 or MAD7 nuclease.

[0182] Genetic construct:

[0183] In an aspect, the invention relates to a genetic construct comprising said exogeneous sequence.

[0184] In an aspect, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms (HA).

[0185] In an aspect, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms, wherein said exogenous sequence comprises • a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon. In an aspect, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0186] In another aspect, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0187] The genetic construct is a nucleotide sequence comprising said exogenous sequence before it is inserted into the genome of a cell.

[0188] The genetic construct may comprise further elements, such as one or more nucleotide sequences, allowing for the targeted insertion, also called knock-in, of said construct or of the exogenous sequence into the genome of the cell.

[0189] Said further elements allowing for targeted insertion, the design and sequence(s) thereof, depend on the mean used for targeted insertion and on the targeted insertion site.

[0190] Targeted insertion may be performed by any known mean or tool for targeted insertion, such tools based on homologous recombination. By way of example, possible means include nuclease based gene editing tools such as TALEN (Transcription activator-like effector nucleases) or RNA-guided nucleases like CRISPR based technologies, such as Cas9 or MAD7 nuclease.

[0191] For targeted insertion by homologous recombination, said further elements may comprise homology arms. In an embodiment, the invention relates to a genetic construct comprising an exogenous sequence flanked by homology arms located on the 5' side and on the 3’ side of said exogenous sequence.

[0192] In an embodiment, each homology arm comprises a nucleotide sequence that is homologous to the sequence of a target insertion site in the genome of a cell.

[0193] In the present invention, said target site is located within the intron of an endogenous gene of interest in a cell.

[0194] In an embodiment, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms, said homology arms comprising one or more sequences homologous to the sequence of a targeted insertion site located within the intron of an endogenous gene of interest.

[0195] In an aspect, the invention relates to a genetic construct comprising said exogenous sequence flanked by homology arms, wherein said homology arms comprise one or more sequences homologous to the sequence of a targeted insertion site located within an intron of an endogenous gene in the genome of the cell.

[0196] Targeted insertion tools are well defined in the art and persons of skill in the art know how to design homology arm sequences adapted to a desired target insertion site and a selected targeted insertion tool.

[0197] In a particular embodiment, the exogenous sequence as defined herein is located within the intron of both alleles of said endogenous gene of interest. This may be referred to as biallelic insertion or biallelic intronic insertion or biallelic targeted intronic insertion (biallelic TH).

[0198] The figure Fig. 1 illustrates some embodiments of targeted insertion of various exogenous sequences (Fig. 1 (E) to (H)) in an intron of an endogenous gene (Fig. 1 (A)) from various genetic constructs (Fig.1 (B) to (D)).

[0199] (A) represents an endogenous gene in a cell genome with at least two exons and an intron and a targeted insertion site (scissors) within the intron. As for most genes, the intron is naturally flanked by a splice donor “SD” and a splice acceptor “SA”.

[0200] (B), (C) and (D) are genetic constructs comprising three different exogenous sequences flanked by homology arms “HA left” and “HA right”, said exogenous sequences comprising respectively (B) only a “shutdown sequence” (“(i) first sequence”), (C) a “shutdown sequence” and a “transgene sequence” (“(ii) second sequence”), (D) a “shutdown sequence” and two “transgene sequences” (“(ii) second sequence” and (iii) third sequence). The skilled person knows that the sequences of the homology arms do not integrate as such, they allow homology directed repair.

[0201] (E), (F), (G), (H) represent the endogenous gene after targeted insertion attempts of (B), (C) and (D).

[0202] (E) represents the endogenous gene after unsuccessful targeted insertion attempt: it shows an “indel” event which is a possible outcome of targeted insertion attempts, whereby the intended targeted insertion has not succeeded.

[0203] (F) represents the endogenous gene after the successful targeted insertion of (B).

[0204] (G) represents the endogenous gene after the successful targeted insertion of (C).

[0205] (H) represents the endogenous gene after the successful targeted insertion of (D).

[0206] The figure Fig. 2 illustrates an example of exogenous sequence comprising two transgene coding sequences, inserted in a cell genome.

[0207] Gene Edit #1 illustrates the biallelic insertion in an intron of endogenous B2M gene (located on chromosome 15) of an exogenous sequence comprising a first sequence per the invention although not illustrated, a second sequence comprising a CAG promoter, a HLA-G (or E) transgene coding sequence, also comprising a stop codon and a polyA sequence although not illustrated, and comprising a third sequence comprising a UBC promoter, a CD55 transgene coding sequence, as well as a stop codon and a polyA sequence although not illustrated.

[0208] The figure Fig. 3 illustrates the targeted insertion of an exogenous sequence in an intron of an endogenous gene, in this case intron 1 of the B2M gene, from a genetic construct comprising an exogenous sequence flanked by homology arms, wherein the exogenous sequence comprises a “shutdown sequence” (“(i) first sequence”). Fig. 3 further shows the situation at the translation level depending on the outcome of the targeted insertion in a given cell:

[0209] (i) “no insertion": if the targeted insertion has not succeeded, the endogenous gene is transcribed to the full mRNA which in turns is translated into the complete endogenous protein, in this case a B2M protein. The cell expresses B2M protein.

[0210] (ii) “monoallelic insertion”: if the targeted insertion has succeeded in one allele of the endogenous gene and not in the other:

[0211] - From the allele with no insertion: a complete mRNA is generated and translated into a complete endogenous protein. The cell expresses B2M protein from the allele without insertion. - From the allele with insertion, mRNAs are generated by the transcription machinery reading the genomic sequence from endogenous exon 1 , continuing over intron 1 , on to the exogenous sequence from the splice acceptor sequence “SA” and up to the first polyA sequence “pA” where mRNAs stop. The exogenous splice acceptor sequence “SA” is involved in the maturation of the mRNAs. The translation machinery generates a truncated protein from the endogenous promoter and exon 1 , stopping shortly after exon 1 , including in this case few amino acids from B2M exon 3”. The truncated protein is not functional and gets degraded in the cell.

[0212] (iii) “biallelic insertion”: if the targeted insertion has succeeded in both alleles of the endogenous gene, a truncated version of the endogenous protein is expressed and gets degraded in the cell. The cell does not express B2M protein.

[0213] Fig. 4A illustrates the targeted insertion of an exogenous sequence in an intron of an endogenous gene, in this case intron 1 of the B2M gene, from a genetic construct comprising an exogenous sequence flanked by homology arms, wherein the exogenous sequence comprises a “shutdown sequence” (“(I) first sequence”) and a “transgene sequence" (“(ii) second sequence”). Fig. 4 further shows the situation at the translation level depending on the outcome of the targeted insertion in a given cell:

[0214] (i) “no insertion”: if the targeted insertion has not succeeded, the endogenous gene is transcribed to the full mRNA which in turn is translated into the complete endogenous protein, in this case a B2M protein. The cell expresses B2M protein.

[0215] (ii) “monoallelic insertion”: if the targeted insertion has succeeded in one allele of the endogenous gene and not in the other:

[0216] - From the allele with no insertion: a complete mRNA is generated and translated into a complete endogenous protein. The cell expresses B2M protein from the allele without insertion.

[0217] - From the allele with insertion, mRNAs are generated by the transcription machinery reading the genomic sequence from endogenous exon 1 , continuing over intron 1 , on to the exogenous sequence from the splice acceptor sequence “SA" and either up to the first polyA sequence “pA” where some mRNAs stop, or continuing over to the remaining of the inserted exogenous sequence up to the second polyA sequence “pA” thereby generating longer mRNAs with the transgene. The exogenous splice acceptor sequence “SA” is involved in the maturation of the mRNAs. From the shorter mRNAs, the translation machinery generates a truncated protein from the endogenous promoter and exon 1 , stopping shortly after exon 1 , including in this case few amino acids from B2M exon 3. The truncated protein is not functional and gets degraded in the cell. From the longer mRNAs, the translation machinery operates from the exogenous promoter and on to the exogenous transgene coding sequence and generates a complete transgenic protein. The cell expresses the transgenic protein.

[0218] (iii) “biallelic insertion”: if the targeted insertion has succeeded in both alleles of the endogenous gene, a truncated version of the endogenous protein is expressed and gets degraded in the cell. The cell does not express B2M protein. The cell expresses a complete transgenic protein from the exogenous transgene coding sequence.

[0219] Fig. 4B is a further illustration of a targeted insertion of an exogenous sequence as disclosed herein. The upper part of Fig.4B entitled “B2M locus” shows an endogenous gene of interest being a B2M gene, a targeted insertion (Tl) locus indicated as position “367” located in intron 1 of said B2M endogenous gene, between exon 1 and exon 2 thereof. The middle part of Fig.4B entitled “B2M locus edited” shows said B2M gene after the targeted insertion in locus “367” of a genetic construct comprising -SA-B2MExon 3-Stop-polyA- sequence followed by - CAG promoter-CD47 transgene-polyA sequence, further followed by EF1a promoter-HLA-E- polyA sequence. The targeted insertion has been mediated by a nuclease and a guide RNA indicated as “guide 367”. The lower part of Fig.4B shows the transcription of the B2M edited gene into a truncated B2M exon1-exon 3 transcript, a functional CD47 transcript and a functional HL!-E transcript. The former non-functional B2M exon1-exon3 transcript will get degraded, thereby acting as a shutdown sequence, and the function C47 and HLA-E transcripts will get translation into corresponding functional proteins.

[0220] In an embodiment, an exogenous sequence as disclosed herein comprises one or more than one transgene sequences. In an embodiment, an exogenous sequence as disclosed herein comprises two transgene sequences.

[0221] Said one or more transgene sequences may be selected from:

[0222] - a transgene sequence comprising a CAG promoter, a B2M-HLA-E coding sequence or a B2M-HLA-G coding sequence, a stop codon, a polyA sequence,

[0223] - a transgene sequence comprising sequence comprises a EF1 a promoter, a B2M-HLA-E coding sequence or a B2M-HLA-G coding sequence, a stop codon, a polyA sequence,

[0224] - a transgene sequence comprising a CAG promoter, a CD47 coding sequence, a stop codon, a polyA sequence,

[0225] - a transgene sequence comprising a EF1a promoter, a CD47 coding sequence, a stop codon, a polyA sequence, - a transgene sequence comprising a CAG promoter, a CD55 coding sequence, a stop codon and a polyA sequence,

[0226] - a transgene sequence comprising a EF1a promoter, a CD55 coding sequence, a stop codon and a polyA sequence,

[0227] - a transgene sequence comprising a CAG promoter, a PD-L1 coding sequence, a stop codon and a polyA sequence,

[0228] - a transgene sequence comprising a EF1a promoter, a PD-L1 coding sequence, a stop codon and a polyA sequence.

[0229] The constructs and exogenous sequences of the invention as described herein bring multiple benefits:

[0230] It makes it possible to achieve both a gene shutdown and a gene knock-in in only one targeted insertion step, which is a significantly simplifies the gene editing process.

[0231] It makes it possible to select the cells that have successfully achieved biallelic insertion of the construct or sequence of interest in only one selection step, based only on the detection of the lack of endogenous protein: cells in which the endogenous protein is detected have not achieved the desired biallelic insertion, whereas cells in which the endogenous protein is not detected have achieved the desired biallelic insertion. The cells may be detected and sorted in one step.

[0232] It makes it possible to achieve very pure biallelic insertion success rate, with a very large proportion of cells subjected to targeted insertion of the genetic construct of the invention successfully achieve biallelic insertion thereof.

[0233] It makes it possible to obtain cell populations wherein the endogenous gene is effectively and reliably shutdown, i.e. with no protein expression from the endogenous gene. It makes it possible to achieve reliable exogenous protein expression in cells and at high expression level thanks to the biallelic insertion of the coding transgene sequence.

[0234] Cell:

[0235] The cell may be at various development stage.

[0236] In an embodiment, the cell is a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC). In another embodiment, the cell is derived from a stem cell and may be at any differentiation stage therefrom. In another embodiment, the cell is a mature cell. In an embodiment, the cell is derived from a pluripotent stem cell, such as any cell type derived from a pluripotent stem cell.

[0237] In an embodiment, the cell is a differentiated cell and is obtained by culturing a pluripotent cell under differentiation conditions, such as in presence of differentiation factors. In another embodiment, the cell is a non-natural cell, such as a differentiated cell obtained by genome engineering of a pluripotent cell.

[0238] The cell may be selected from the list consisting of:

[0239] - a beta cell, an INS+ and NKX6.1 + double positive cell or a C-peptide+ / NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+ / NKX2.2+ double positive cell,

[0240] - a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell,

[0241] - an exosome cell,

[0242] - an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell,

[0243] - a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a cardiomyocyte, a retinal cell, a retinal pigment epithelium cell

[0244] - a cortical neural progenitor cell,

[0245] - a mesenchymal stem cell,

[0246] - an hematopoietic stem cell (HSC),

[0247] - multi potent stem cells such as hematopoietic stem cells (HSC),

[0248] - differentiated hematopoietic cells such as T cells, macrophages, NK cells.

[0249] Stem cell:

[0250] As used herein, the term “stem cell” is to be understood as an undifferentiated cell having differentiation potency and proliferative capacity, particularly self-renewal competence, but maintaining differentiation potency. The term “stem cell” includes subpopulations such as pluripotent stem cell (PSC), multipotent stem cell, unipotent stem cell and the like according to the differentiation potency.

[0251] Pluripotent stem cell, also known as pluripotent cell, or pluripotent SC, or PSC: As used herein, these terms refer to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to three germ layers (ectoderm, mesoderm, endoderm). A PSC can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the PSC include embryonic stem cell (ESC), induced pluripotent stem cell (iPSC), embryonic germ cell (EG cell) and the like. Muse cell (Multi-lineage differentiating stress enduring cell) obtained from mesenchymal stem cell (MSC), and germline stem cell (GS cell) produced from reproductive cell (e.g., testis) are also encompassed in the PSC term. The pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03 / 055992 and WO 2007 / 042225, or be commercially available cells or cell lines. ES cell lines can also be derived from single blastomeres without the destruction of ex utero embryos and without affecting the clinical outcome (Chung et al. (2006) and Klimanskaya et al. (2006)). Embryonic stem cells may also be derived from parthenotes as described in e.g. WO 2003 / 046141 . Additionally, embryonic stem cells can be produced from a single blastomere or by culturing an inner cell mass obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available.

[0252] Induced pluripotent stem cell, iPS, IPSCs:

[0253] As used herein, the term “induced pluripotent stem cell” (also known as iPS cells or IPSCs) means a type of PSC that can be generated directly from adult cells by a process commonly known as reprogramming. By the introduction of products of specific sets of pluripotency- associated genes adult cells can be converted into PSCs

[0254] Preferably, the methods and products of the present invention are based on hPSCs, i.e. stem cells derived from either iPSCs or embryonic stem cells, including parthenotes.

[0255] Differentiated cells:

[0256] As used herein, the term “differentiated cell” means a cell which does not have the potency to differentiate into any cell lineage. Differentiated cells may be obtained from stem cells or the like upon exposure to appropriate differentiation culture conditions. Differentiated cells may be obtained from donors.

[0257] Endocrine progenitor cell:

[0258] As used herein, the term “endocrine progenitor cell” refers to a cell characterised by expression of markers NGN3, NeuroD and NKX2.2. As used herein, the term “NGN3+ / NKX2.2+ double positive cell” refers to a cell that co-express the two markers NGN3 and NKX2.2. As used herein, the term “NeuroD” refers to a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors, the term "NGN3" refers to a member of the neurogenin family of basic loop- helix-loop transcription factors, and the terms “NKX2.2" and "NKX6.1" refer to members of the NKX transcription factor family.

[0259] An “INS+” cell as used herein is a cell that produces insulin.

[0260] The terms “differentiation” or “cell differentiation”, “differentiating”, as used herein refer to cellular differentiation. Cellular differentiation is the process in which a cell changes from one cell type to another, typically from a less specialized type, such as a stem cell, to a more specialized type, such as a tissue specific cell, e.g. a cardiomyocyte. The terms “differentiated” and “undifferentiated” refer to the stage of differentiation of a cell in the cellular differentiation process.

[0261] In a preferred embodiment, the cell is a stealth cell, also called immune protected cell. The terms “stealth” or “immune protected” cell designate a genetically modified cell such that it is less or no longer susceptible of rejection by the immune system of a host upon allogeneic transplantation of said cell compared to the same cell without said genetic modification. Such property is particularly beneficial in the context of cellular therapy. It may be achieved by the disruption of genes involved in immune recognition and / or rejection mechanisms, such as B2M gene, CIITA gene, TRAC gene, HLA-A gene, HLA-B gene, HLA-C gene, CD155 gene, CD122 gene and B7-H3 gene. Disruption of anyone of these genes may be obtained by the biallelic insertion of an exogenous sequence as described herein within an intron thereof (shutdown), or by deletion thereof or a further disruption method.

[0262] In an embodiment, the cell is B2M-Z-. In a specific embodiment, the cell is B2M- / - and comprises the biallelic insertion of an exogenous sequence as described herein within an intron, such as intron 1 , of said cell’s endogenous B2M gene.

[0263] In an embodiment, the cell is CIITA- / -. In a specific embodiment, the cell is CIITA- / - and comprises the biallelic insertion of an exogenous sequence as described herein within an intron, such as intron 1 , of said cell’s endogenous CIITA gene.

[0264] As used herein, the term “- / -" referring to a specific gene, i.e. “gene - / -“ such as in exemplary expressions “B2M - / -“, “CIITA - / -“ etc., means that both alleles of said genes are deficient in a given cell.

[0265] In an embodiment, the cell comprises a B2M-HLA genetic fusion transgene. In an embodiment, said B2M-HLA transgene is a transgene coding sequence comprised in the exogenous sequence of the present invention. In an alternative embodiment, said B2M-HLA transgene is inserted in the cell’s genome independently of the exogenous sequence of the present invention.

[0266] In a preferred embodiment, the cell is B2M- / -, CIITA- / - and / or comprises a B2M-HLA genetic fusion transgene. In an embodiment, said B2M-HLA genetic fusion transgene is B2M-HLA-E and / or B2M-HLA-G.

[0267] In an embodiment, the cell comprises an exogenous inserted in an intron of an endogenous gene of interest and in both alleles thereof, wherein the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence comprising exon 3 or a portion thereof of human B2M gene, such as sequence SEQ ID NO 4 or a portion thereof, a stop codon, a 3’UTR sequence or a portion thereof and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence comprising a B2M-HLA fusion gene, such as B2M-HLA-E fusion gene and / or B2M- HLA-G fusion gene, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence; and wherein the endogenous sequence is B2M endogenous gene, and wherein said exogenous sequence is inserted in intron 1 or 2, such as intron 1 , of said endogenous gene of interest and in both alleles thereof.

[0268] The cells may additionally be engineered to have reduced or increased expression of additional immune evasive proteins on the cell surface. These could be, but is not limited to, PDL1 , complement factors like, CD47, CD55, CD46, CD59, CD35, and / or other immune evasive ligands, or analogues thereof.

[0269] A cell may comprise more than one targeted intronic insertion of exogenous sequence.

[0270] A cell may comprise two or more exogenous sequences, inserted in two or more different endogenous genes, in a intron thereof.

[0271] A cell may comprise an exogenous sequence as defined herein within an intron of an endogenous gene of interest and in both alleles thereof, a further exogenous sequence located within an intron of another endogenous gene of interest and in both alleles thereof, and potentially one or more further exogenous sequences, each located within an intron of a further endogenous gene of interest and in both alleles thereof.

[0272] Cell population In an aspect, the invention relates to a cell population comprising cells as defined herein. In an aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence as defined herein.

[0273] In an aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises a sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0274] In an aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0275] In another aspect, the invention relates to a cell population comprising cells, said cells comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises:

[0276] I. a first sequence comprising a splice acceptor sequence, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0277] In an embodiment, the invention relates to a cell population comprising cells, wherein at least 93% such as at least 94%, 95%, 96%, 97% or 98% of the cells comprised in said population are cells as defined herein.

[0278] In an embodiment, the invention relates to a cell population comprising cells, wherein at least 98%, and preferably wherein at least 99%, at least 99,5%, at least 99,8% and even more preferably 100% of the cells comprised in said population are cells comprising said exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof.

[0279] In an embodiment, the cell population comprises cells, wherein at least 93% of said cells comprise an exogenous inserted in an intron of an endogenous gene of interest and in both alleles thereof, wherein the exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence comprising exon 3 of human B2M gene or a portion thereof, such sequence SEQ ID NO 4 or a portion thereof, a stop codon, a 3’UTR sequence or a portion thereof and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence comprising a B2M-HLA fusion gene, such as B2M-HLA-E fusion gene and / or B2M- HLA-G fusion gene, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence; and wherein the endogenous sequence is B2M endogenous gene, and wherein said exogenous sequence is inserted in intron 1 or 2, such as in intron 1 , of said endogenous gene of interest and in both alleles thereof.

[0280] In an aspect, the invention relates to a pharmaceutical composition comprising cells as defined herein and a pharmaceutically acceptable excipient.

[0281] In another aspect, the invention relates to a pharmaceutical composition comprising a cell population a pharmaceutically acceptable excipient, wherein at least 98%, and preferably wherein at least 99%, at least 99,5%, at least 99,8% and even more preferably 100% of the cells comprised in said population are cells comprising said exogenous sequence as defined herein located within an intron of an endogenous gene of interest and in both alleles thereof.

[0282] Each element of said cells, said exogenous sequence, said intro and said endogenous gene is as defined herein. Method of manufacturing

[0283] In an aspect, the invention relates to a method of manufacturing a cell population as defined herein.

[0284] Figure 14 schematically illustrates the invention and figures 1 1 to 13 illustrate alternative editing approaches.

[0285] Figure 11 schematically illustrates exon editing of a cell surface protein using Non Homologous End Joining (NHEJ). As starting material, an unedited cell (indicated as (a) in Fig.11) expresses the target protein on its cell surface. After genome editing with a DNA double-strand break (DSB)-inducing nuclease in the target protein's exon sequence, the DSB is repaired by nonhomologous end-joining (NHEJ) in absence of a donor DNA. Only bi- allelic edited cells (red, indicated as (b) in Fig.11) will lose expression of the target protein and can be enriched by FACS against unedited cells (black, (a)). NHEJ creates random indels at the target site. Therefore, enriched shutdown clones will be heterogenous ((b) in Fig.11) for the shutdown-causing mutation. Any transgene needs to be inserted in sequential second edit and at a different target site.

[0286] As illustrated in Fig.11 B, with exon editing of a cell surface protein using NHEJ, direct enrichment for bi-allelic shutdown results in a pool of cells ((b) in Fig.11) comprising many different mutations, and any transgene needs to be added in a second editing round.

[0287] Figure 12 schematically illustrates exon editing of cell surface protein and transgene insertion using Homology-Directed Repair (HDR). As starting material, an unedited cell (indicated as (a)) expresses the target protein on its cell surface. After genome editing with a DSB- inducing nuclease in the target protein's exon sequence the DSB is repaired by homology- directed repair (HDR) in presence of a donor DNA. Bi-allelic shutdown of the target protein can be the result of bi-allelic targeted transgene insertion (TTI) causing a bi-allelic shutdown of the target protein (green, indicated as (c) in Fig.12), mono-allelic TTI in combination with NHEJ-mediated shutdown of the second allele (blue, also indicated as (c) in Fig.12) or bi- allelic NHEJ-driven shutdown, but without TTI (red, indicated as (d) in Fig.12). Insufficient shutdown of the target protein (blue, indicated as (b) in Fig.12) can be the result of mono- allelic TTI. It is not possible to FACS-enrich the target cells (green, indicated as (c), bi-allelic shutdown of the target protein) directly.

[0288] As illustrated in Fig. 12B, with exon editing of cell surface protein and transgene insertion using Homology-Directed Repair (HDR), a pool of cells comprising both shutdown of target protein expression and transgene mono- or bi-allelic Tl is obtained, and it is not possible to enrich directly for bi-allelic Tl, i.e. it is not possible to sort a pool of cells comprising both shutdown of target protein expression and bi-allelic-only transgene Tl.

[0289] Figure 13 schematically illustrates intron editing of cell surface protein using NHEJ. As starting material, an unedited cell (indicated as (a)) expresses the target protein on its cell surface. NHEJ-based genome editing in the intron sequence of the target protein in absence of any donor DNA does not lead to any functional shutdown. Unedited (indicated as (a)) and edited (indicated as (b)) cells will show ongoing expression of the target protein (black, (a) and (b)).

[0290] As illustrated in Fig. 13B, with intron editing of cell surface protein using NHEJ, it is not possible to generate any shutdown comprising cell, and any transgene needs to be added in a second editing round.

[0291] Figure 14 schematically illustrates intron editing of cell surface protein and transgene insertion using HDR. As starting material, an unedited cell (indicated as (a)) expresses the target protein on its cell surface. HDR-based TTI in the intron sequence of the target protein in presence of a donor DNA does lead to shutdown of the target protein in a bi-allelic fashion only (green, indicated as (b)). These target cells can be enriched directly and at high purity by FACS. Mono-allelic TTI (blue, indicated as “(c) monoallelic Tl” in Fig.14) and NHEJ- repaired cells (black, indicated as “(c) no Tl”) still express the target protein and can be distinguished by FACS from the target cells (green, indicated as (b)). The donor requires 5’UTR so-called shutdown sequence (lila, indicated with * in Fig.14A) to drive shutdown from an intronic insertion.

[0292] As illustrated in Figure 14B, with intron editing of cell surface protein and transgene insertion using HDR, it is possible to sort a pool of cells comprising both shutdown of protein expression and Tl of transgene (bi-allelic, indicated as (b) in Fig.14) and direct enrichment of target cells (b) is obtained. Intron-TI mediated shutdown requires a shutdown sequence (lila, *)■

[0293] As illustrated in Figure 14, intron-TI (“intron Tl” stands for “targeted insertion into an intron) mediated shutdown allows to generate in one step and to sort in one step a cell population comprising both shutdown of a target protein expression, and bi-allelic targeted insertion of a transgene of interest. This is made possible by the genetic construct as described herein. The construct and / or method of the invention allow for direct selection of cellular clones that carry the bi-al lei ic targeted insertion of a transgene of interest as well as the bi-al lelic targeted shutdown of a targeted endogenous gene of interest, without clones comprising mono-allelic targeted insertion of said transgene of interest and / or mono-allelic shutdown of said targeted endogenous gene of interest.

[0294] As illustrated in Figures 11 to 13, none of exon editing of a cell surface protein using NHEJ, exon editing of cell surface protein and transgene insertion using Homology-Directed Repair (HDR) and intron editing of cell surface protein using NHEJ allow for direct selection of cellular clones that carry the bi-allelic targeted insertion of a transgene of interest as well as the bi-allelic targeted shutdown of a targeted endogenous gene of interest.

[0295] In an aspect, the invention relates to a method of manufacturing a cell population, said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of an exogenous sequence as defined herein.

[0296] In an embodiment, in said gene editing step, said insertion is targeted in an intron of an endogenous gene of interest in the cells of said cell population. This targeted insertion may be achieved by homologous recombination. Homologous recombination may be achieved with homology arms flanking said exogenous sequence, said homology arms comprising one or more sequences homologous to the sequence of said targeted intron.

[0297] In an aspect, the invention relates to a method of manufacturing a cell population, said manufacturing method comprises: a gene editing step wherein a cell population is subjected to the targeted insertion of a genetic construct as defined herein.

[0298] In an embodiment, said genetic construct comprises an exogenous sequence as defined herein flanked by homology arms. Said homology arms comprise one or more sequences homologous to a targeted sequence in an intron of an endogenous gene of interest in the cells.

[0299] Said homology arms, said intron, said endogenous sequence, said exogenous sequence and their components, and said cells are as defined herein. In an aspect, the invention relates to a method of manufacturing a cell population, said manufacturing method comprises: a gene editing step wherein a cell population is subjected to the targeted insertion of a genetic construct, said genetic construct comprising an exogenous sequence as defined herein flanked by homology arms, wherein said homology arms comprise one or more sequences homologous to the sequence of a targeted insertion site located within an intron of an endogenous gene in the genome of said cells.

[0300] In an embodiment, upon said gene editing step, the cells in said cell population comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene.

[0301] In an embodiment, the method of manufacturing of a cell population comprises: a gene editing step wherein a cell population is subjected to targeted insertion of an exogenous sequence in an intron of an endogenous gene in the cells, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a coding sequence comprising human B2M gene exon 3 or a portion thereof, such as a portion of sequence SEQ ID NO 4 , a stop codon, a 3’UTR sequence or a portion thereof and a polyA sequence, and ii. a second sequence comprising a promoter, a transgene coding sequence comprising a B2M-HLA fusion gene, such as B2M-HLA-E fusion gene and / or B2M- HLA-G fusion gene, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence; and wherein said endogenous sequence is B2M endogenous gene.

[0302] In a particular embodiment, said exogenous sequence is inserted in intron 1 or 2, such as intron 1 of said endogenous gene of interest and in both alleles thereof.

[0303] In an embodiment, in said gene editing step, said cell population is subjected to targeted insertion of two or more genetic constructs or exogenous sequences as defined herein, wherein each of said genetic constructs or exogenous sequences targets a different endogenous gene of interest. In an embodiment, said cell population is subjected to the targeted insertion of two or more genetic constructs, sequentially.

[0304] Each element of said cells, said genetic construct, said exogenous sequence, said homology arms and / or said endogenous gene etc. are as defined herein.

[0305] In an aspect, the invention relates to a method of manufacturing a cell population, said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct or exogenous sequence as defined herein; and a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous gene of interest.

[0306] In an aspect, the invention relates to a method of manufacturing a cell population, said manufacturing method comprises:

[0307] (a) a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined herein; and

[0308] (b) an enrichment step comprising:

[0309] (b) (i) a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest; and

[0310] (b) (ii) a sorting step wherein cells in which no presence of the protein encoded by said endogenous of interest is detected are selected, and / or cells in which presence of the protein encoded by said endogenous of interest is detected are deselected, wherein said detection (b)(i) and sorting (b)(ii) steps may be performed successively or concomitantly.

[0311] In a preferred embodiment, the cell population obtained upon said enrichment step or said sorting step comprises at least 93% of the cells in said cell population, such as at least 94%, 95%, 86%, 97% or 98% of the cells in the cell population comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest.

[0312] In a preferred embodiment, the cell population obtained upon said enrichment step or said sorting step comprises at least 98% of the cells in said cell population, preferably at least 99%, at least 99,5%, at least 99,8% and even more preferably 100% of the cells in the cell population resulting from said gene editing step comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest.

[0313] In an embodiment, the method of manufacturing subject of the present invention is a method of manufacturing a cell population, wherein at least 93% of the cells in said cell population, such as at least 94%, 95%, 86%, 97% or 98% of the cells in said cell population, such as at least 99%, at least 99,5%, at least 99,8% or 100% of the cells in said cell population comprise the biallelic insertion of an exogenous sequence within an intron of an endogenous gene of interest.

[0314] It was surprisingly discovered that the genetic constructs and exogenous sequences of the invention allow a particularly high targeted insertion efficacy.

[0315] It was surprisingly discovered that the genetic constructs and exogenous sequences of the invention allow a particularly high sorting and enrichment efficacy.

[0316] This is particularly advantageous as it saves significant amount of time and resources in the manufacturing of gene edited cells. In situations where multiple genes are to be edited, such as multiple endogenous genes disruptions and transgenes insertions, the present invention greatly simplifies the manufacturing process or even enables the manufacturing of gene edited cells of interest. This is particularly useful in the field of cell therapy where numerous gene editings are necessary and particularly difficult to achieve. This is also particularly useful in the field of cell therapy where safety and / or efficacy of a therapeutic cell product require total absence of certain endogenous proteins, such as B2M, CIITA and / or some HLA proteins.

[0317] In an embodiment, said detection step is performed by FACS with an antibody to the protein encoded by said endogenous gene of interest, or to a cell surface protein which requires the protein encoded by said endogenous gene of interest for its translocation to the cell surface or otherwise for its presence on the cell surface. In an embodiment, the detection step (b)(i) and the sorting step (b)(ii) are performed concomitantly such that they may be considered as being performed as one same step, typically in a cell sorting apparatus which detects and sorts accordingly, such as by FACS. In an embodiment, said enrichment step is performed by FACS.

[0318] In an embodiment, the detection and sorting step (b)(i) of the presence and / or absence of the protein encoded by the endogenous gene of interest is performed directly, for example using a compound, such as an antibody, that binds specifically to said protein.

[0319] In an embodiment, the detection step (b)(i) of the presence and / or absence of the protein encoded by the endogenous gene of interest is performed indirectly. In this case, the detection step is performed on another protein, the detection of which is dependent on the presence or absence of the protein encoded by the endogenous gene of interest.

[0320] By way of example, if the endogenous gene of interest is the B2M gene, the detection step may be the detection of a HLA-I protein on the cell(s) surface, because HLA-I proteins require the presence of a B2M protein to translocate to the cell surface. Hence the absence or presence of HLA-I protein on the cell surface directly correlates with the absence or presence of B2M protein expression in the cell.

[0321] The protein subject of the detection step may be an intracellular protein or a cell surface protein. In a preferred embodiment, the protein subject of the detection step is a cell surface protein.

[0322] In an embodiment, the sorting step (b)(ii) selects cells in which no protein encoded by the endogenous gene of interest is detected. These are cells that comprise the biallelic insertion of an exogenous sequence as defined herein within an intron of an endogenous gene of interest.

[0323] In an embodiment, the enrichment step (b) results in a cell population enriched in cells in which no protein encoded by the endogenous gene of interest is detected, i.e. enriched in cells comprise the biallelic insertion of an exogenous sequence as defined herein within an intron of an endogenous gene of interest.

[0324] In an embodiment, the enrichment step results in a cell population comprising at least 93%, such as at least 94%, 95%, 86%, 97% or 98%, or 99% at least 99,5%, at least 99,8% or 100% of the cells, comprising the biallelic insertion of an exogenous sequence as defined herein within an intron of an endogenous gene of interest. In an embodiment, the invention relates to a method of manufacturing a cell population comprising:

[0325] (a) a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct or exogenous sequence as defined herein; and

[0326] (b) an enrichment step comprising:

[0327] (b) (i) a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest, directly or indirectly; and

[0328] (b) (II) a sorting step wherein cells are selected in which no presence of the protein encoded by said endogenous of interest is detected, directly or indirectly, wherein an enriched cell population is obtained and at least 93%, such as at least 94%, 95%, 86%, 97% or 98%, or 99% at least 99,5%, at least 99,8% or 100% of the cells in said enriched cell population comprise the biallelic targeted insertion of said exogenous sequence.

[0329] The present invention significantly facilitates the sorting of cells upon transfection.

[0330] It allows cells sorting by negative selection of cells of interest, i.e. by sorting of cells based on the absence of the protein normally expressed by the endogenous gene of interest. Such negative selection requires no cell treatment for selection. If the protein normally expressed by the endogenous gene of interest is a cell surface protein or is necessary for the presence of another protein on the cell surface, the sorting only requires an antibody to said cell surface protein.

[0331] The manufacturing process based on the genetic construct of the invention advantageously allows to sort out in only one step the cells comprising both the biallelic shutdown of an endogenous gene of interest, hence the complete disruption of expression of said gene, and the biallelic insertion of a transgene of interest. Indeed, in a cell comprising a biallelic insertion of the genetic construct as defined herein at the desired insertion target site located in an intron of an endogenous gene of interest, both alleles of said endogenous gene are disrupted such that the protein normally expressed by said gene is no longer present in the cell. Therefore, by detecting the absence of said protein, whether directly or indirectly, it is possible to select in one same step the cells of interest, i.e. the cells in which no presence of said protein is detected, because these cells necessarily have both alleles of said endogenous gene successfully disrupted and two copies of said genetic construct successfully inserted.

[0332] The term “biallelic insertion” of a genetic construct within an endogenous gene of interest means insertion of said genetic construct within both alleles of said endogenous gene. The term “biallelic disruption” or “biallelic shutdown” of a gene means disruption of both alleles of said gene.

[0333] The detection step and sorting step may be performed by any means known in the art, any technique allowing to detect proteins on / in cells and / or to sort cells.

[0334] For the detection of protein on the cell surface, applicable techniques known in the art include fluorescence activated cell sorting (FACS), magnetic beads cells sorting (MACS), living cell chromatography, column sorting.

[0335] The present invention allows a significantly improved cell editing and sorting process. Compared to PCR-based single clone selection, which is a lengthy process typically over several weeks, requires single clone multiplication into clonal population and replica thereof and leads to low cells survival rate, such as around 50%, the present invention in contrast allows for much faster and efficient cell editing and sorting process: the present invention allows to screen in one same enrichment step, for example by FACS, the cells lacking the targeted (i.e. shutdown) endogenous protein, thereby obtaining a cell population comprising only the cells that have successfully inserted the desired biallelic edits, comprising one or even several transgenes. With the present invention, it typically takes an hour to sort 50000 cells.

[0336] In an embodiment, upon said enrichment step, at least 93%, such as at least 94%, 95%, 96%, 97% or 98% of the cells in said cell population, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene, i.e. comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest.

[0337] In an embodiment, upon said enrichment step, at least 99,9% and preferably 100% of the cells in said cell population, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene, i.e. comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest. In an embodiment, said method of manufacturing comprises no more than one enrichment step.

[0338] In an embodiment, said method of manufacturing comprises no more than one enrichment step per gene editing step.

[0339] In an embodiment, said method of manufacturing comprises no more than one enrichment step per genetic construct i.e. per targeted endogenous gene.

[0340] In an embodiment, said method of manufacturing comprises the targeted insertion of two or more genetic constructs into different endogenous genes. In this case, the detection of the presence or absence of proteins encoded by said endogenous genes may be performed directly or indirectly on all proteins in the same detection step or in successive detection steps.

[0341] In an aspect, the invention relates to a cell population obtained by a method of manufacturing as defined herein. In an aspect, the invention relates to a cell obtained by a method of manufacturing as defined herein.

[0342] In an aspect, the invention relates to the use of a genetic construct as defined herein for the manufacturing of a cell population wherein at least 98% of the cells, such as at least 99%, at least 99,5%, at least 99,8% and even more preferably 100% of the cells present in said cell population comprise both the biallelic insertion of said genetic construct and the biallelic shutdown of said endogenous gene of interest.

[0343] In an aspect, the invention relates to a cell of the invention or a cell population as defined herein is for use as a medical treatment.

[0344] In an embodiment, said medical treatment is a cell therapy.

[0345] In an embodiment, cell therapy is cell replacement therapy.

[0346] Cell therapy, such as cell replacement therapy, is applicable in the treatment of a large variety of diseases.

[0347] In an embodiment, the cell, cell population and / or pharmaceutical composition as defined herein is for use in cellular therapy of a patient in need thereof.

[0348] In an embodiment, the use in cell therapy is for the treatment, the cure, or the prevention of a chronic disease or of an acute disease. In an embodiment, the chronic disease is selected from diabetes, type 1 diabetes, type 2 diabetes, dry macular degeneration, retinitis pigmentosa, neurological disease, Parkinson’s disease, heart disease, tissue fibrosis, cirrhosis, hearing loss, corneal blindness, stroke, chronic heart failure, chronic kidney disease or cancer. In an embodiment, the acute disease is selected from bacterial lung infections, such as ventilator acquired bacterial pneumonia or hospital acquired bacterial pneumonia.

[0349] In an embodiment, cell therapy is CAR-T cell therapy.

[0350] In an embodiment, the cell, cell population and / or pharmaceutical composition as defined herein is for use in CAR-T cell therapy of a patient in need thereof.

[0351] In an embodiment, the use in CAR-T cell therapy is for the treatment, the cure, or the prevention of cancer diseases.

[0352] In an aspect, the invention relates to a genetic construct as defined herein is for use as a medical treatment.

[0353] In an embodiment, said medical treatment is gene therapy.

[0354] Non limiting embodiments of the present invention:

[0355] 1 . A cell comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises

[0356] • a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0357] 2. A cell according to embodiment 1 , wherein said exogenous sequence comprises

[0358] • a first sequence comprising a splice acceptor (SA) sequence, a stop codon such as a Tristop sequences and preferably two or more stop codons such as two successive Tristop sequences, a coding sequence and two or more successive polyA sequences, such as three or more, and preferably four or more polyA sequences, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequence(s).

[0359] 3. A cell according to embodiment 1 or 2, wherein said exogenous sequence comprises

[0360] • a first sequence comprising a splice acceptor (SA) sequence, two successive triple stop sequences, a coding sequence which comprises exon 3 of B2M gene or a portion thereof, four successive polyA sequences, such as polyA sequence derived from SV40, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequences. 4. A cell according to embodiment 1 comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises i. a first sequence (shutdown sequence) comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence (transgene sequence) comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences (transgene sequences), each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0361] 5. A cell according to embodiment 4 comprising two or more exogenous sequences located within an intron of different endogenous genes of interest and in both alleles thereof.

[0362] 6. A cell according to embodiment 5 comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, a further exogenous sequence located within an intron of another endogenous gene of interest and in both alleles thereof, and potentially one or more further exogenous sequences located within an intron of one or more other endogenous genes of interest and in both alleles thereof.

[0363] 7. A cell according to embodiment 4, 5 or 6 wherein i. said first sequence comprises a splice acceptor sequence, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon and a polyA sequence.

[0364] 8. A cell according to anyone of the preceding embodiments, wherein the splice acceptor sequence is or comprises anyone of SEQ ID NO 01 , sequence SEQ ID NO 02, sequence SEQ ID NO 03 and sequence AG.

[0365] 9. A cell according to anyone of the preceding embodiments, wherein the coding sequence in the first sequence (shutdown sequence) comprises between 1 to 50 nucleotides, preferably 1 to 40, 1 to 30, 1 to 20, 5 to 20 nucleotides.

[0366] 10. A cell according to anyone of the preceding embodiments, wherein the coding sequence in the first sequence is or comprises human B2M gene exon 3 or a portion thereof , such as SEQ ID NO 4.

[0367] 11. A cell according to anyone of the preceding embodiments, wherein the 3’UTR sequence is or comprises a sequence of TP53 3’UTR such as sequence SEQ ID NO 05. 12. A cell according to anyone of the preceding embodiments, wherein the stop codon(s) are selected, independently of each other, from sequences TAA, TAG, TGA, and one, two or more triple frame stop sequences SEQ ID NO 06.

[0368] 13. A cell according to anyone of the preceding embodiments, wherein said polyA sequence, or said polyA sequences independently of each other, comprise or are selected from SV40 polyA sequence such as SEQ ID NO 07, bGH polyA sequence such as SEQ ID NO 08, sequence SEQ ID NO 09, a synthetic polyA sequence such as SEQ ID NO 10 and sequences derived therefrom and combinations thereof.

[0369] 14. A cell according to anyone of the preceding embodiments, wherein anyone of said polyA sequence comprises more than one polyA sequence, such as 2, 3 or 4 polyA sequences.

[0370] 15. A cell according to embodiment 14, wherein anyone of said polyA sequence comprises 2 SV40 polyA sequences or 4 SV40 polyA sequences.

[0371] 16. A cell according to embodiment 1 , wherein anyone of said polyA sequence is or comprises 2 bGH polyA sequences.

[0372] 17. A cell according to embodiment 14, wherein anyone of said polyA sequence is or comprises 4 bGH polyA sequences.

[0373] 18. A cell according to anyone of the preceding embodiments, wherein said promoter is selected from or said promoters are independently of each other selected from the list consisting of a CAG (CMV early enhancer chicken beta actin) promoter, a UBC (Ubiquitin C) promoter and EF1-alpha (human elongation factor-1 alpha) promoter.

[0374] 19. A cell according to embodiment 18, wherein, said promoter(s) comprises sequence SEQ ID NO 1 1 , SEQ ID NO 12 or SEQ ID NO 13.

[0375] 20. A cell according to anyone of the preceding embodiments, wherein said transgene coding sequence is selected from a T-cell receptor encoding sequence, a CD55 encoding sequence, a PDL1 encoding sequence, a CD47 encoding sequence, a HLA-E encoding sequence, a HLA-F encoding sequence, a B2M-HLA fusion encoding sequence, such as such as a B2M-HLA-E and / or a B2M-HLA-G fusion protein encoding sequence, such as B2M / HLA-E*0101 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein optionally with a (G4S)4 linker and a signal peptide or a B2M / HLA-E*0103 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein optionally with a (G4S)4 linker and a signal peptide, and a suicide switch encoding gene, and analogues thereof and combinations thereof.

[0376] 21 . A cell according to anyone of the preceding embodiments, wherein said exogenous sequence further comprises:

[0377] - a chromatin opening element such as UCOE such as sequence SEQ ID NO 20, - a ribosome skipping element, such as T2A element and / or F2A element, such as sequence SEQ ID NO 19 and / or SEQ ID NO 20, and / or

[0378] - an insulator element, such as cHs4 insulator sequence such as comprising sequence SEQ ID NO 21 or such as Smar 4 buffer sequence such as comprising sequence SEQ ID NO 22, or combinations thereof. A cell according to any one of the preceding embodiments, wherein said cell is a human cell, such as a genetically engineered human cell. A cell according to any one of the preceding embodiments, wherein said cell is a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC). A cell according to any one of embodiments 1-22, wherein said cell is derived from a stem cell and may be at any differentiation stage therefrom. A cell according to any one of embodiments 1-22 and 24, wherein said cell is a mature cell. A cell according to any one of embodiments 1-22 and 24-25, wherein said cell is derived from a pluripotent stem cell, such as any cell type derived from a pluripotent stem cell. A cell according to any one of embodiments 1-22 and 24-26, wherein said cell is a differentiated cell and is obtained by culturing a pluripotent cell under differentiation conditions, such as in presence of differentiation factors. A cell according to any one of embodiments 1-22 and 24-26, wherein said cell is a nonnatural cell, such as a differentiated cell obtained by genome engineering of a pluripotent cell. The cell according to any one of embodiments 1-22 and 24-26, wherein said cell is selected from the list consisting of:

[0379] - a beta cell, an INS+ and NKX6.1+ double positive cell or a C-peptide+ / NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+ / NKX2.2+ double positive cell,

[0380] - a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell,

[0381] - an exosome cell,

[0382] - an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell,

[0383] - a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a cardiomyocyte, a retinal cell, a retinal pigment epithelium cell

[0384] - a cortical neural progenitor cell,

[0385] - a mesenchymal stem cell,

[0386] - an hematopoietic stem cell (HSC),

[0387] - multipotent stem cells such as hematopoietic stem cells (HSC), and

[0388] - differentiated hematopoietic cells such as T cells, macrophages, NK cells.

[0389] 30. A cell according to anyone of the preceding embodiments, wherein said endogenous gene is selected from the list consisting of B2M ( 2 microglobulin) gene, CIITA (class II, major histocompatibility complex transactivator) gene, TRAC (T-cell receptor alpha constant) gene, HLA-A gene, HLA-B gene, HLA-C gene, CD 155 (also known as poliovarius receptor or PVR) gene, CD122 (also known as interleukine-2 receptor subunit beta or IL2RB) gene and B7-H3 (B3 homolog 3, also known as cluster of differentiation 276 or CD276) gene and TCR-gene.

[0390] 31 . A cell according to anyone of the preceding embodiments, wherein said endogenous gene is the B2M ([32 microglobulin) gene, said exogenous sequence comprises a HLA-G or HLA-E transgene coding sequence and a CD55 transgene coding sequence.

[0391] 32. A cell according to embodiment 30, wherein said HLA-G or HLA-E transgene coding sequence is under the control of a CAG promoter and said CD55 transgene coding sequence is under the control of a EF1 a promoter.

[0392] 33. A cell according to anyone of the preceding embodiments, wherein said first sequence comprises a splice acceptor, a B2M exon 3 coding sequence, a 3’UTR sequence, and four SV40 polyA sequences.

[0393] 34. A cell according to embodiment 32, wherein said endogenous gene is the B2M (02 microglobulin) gene, said exogenous sequence comprises a HLA-G or HLA-E transgene coding sequence and comprises a CD55 transgene coding sequence, wherein said transgene coding sequences are separately located in the second or the further sequence.

[0394] 35. A cell according to embodiment 33, wherein said HLA-G or HLA-E transgene coding sequence is under the control of a CAG promoter and said CD55 transgene coding sequence is under the control of a EF1 a promoter.

[0395] 36. A cell according to embodiments 33 or 34, wherein

[0396] • said second or further sequence comprises the sequence UCOE-CAG(promoter)-B2M- HLAE0103 with peptide (transgene)-stop codon-polyA sequence and wherein

[0397] • said further or second sequence comprises the sequence EF1a (promoter)-CD55 (transgene)-stop codon-polyA sequence. 37. A cell according to anyone of embodiments 30-36, wherein said endogenous gene is the B2M (p2 microglobulin) gene, said exogenous sequence comprises a B2M-HLA-G or B2M-HLA-E transgene coding sequence and a CD47 transgene coding sequence.

[0398] 38. A cell according to embodiment 37, wherein said B2M-HLA-G or B2M-HLA-E transgene coding sequence is under the control of a EF1 a promoter and said CD47 transgene coding sequence is under the control of a CAG promoter.

[0399] 39. A cell according to anyone of embodiments 30, wherein said endogenous gene is the CIITA gene, said exogenous sequence comprises a PD-L1 transgene coding sequence and comprises a CD55 transgene coding sequence.

[0400] 40. A cell according to embodiment 39, wherein said PD-L1 transgene coding sequence is under the control of a CAG promoter and said CD55 transgene coding sequence is under the control of a EF1 a promoter.

[0401] 41 . A cell population comprising cells as defined in any one of the preceding embodiments.

[0402] 42. A cell population according to embodiment 41 , wherein at least 93% such as at least 94%, 95%, 96%, 97% or 98% of the cells comprised in said population are cells as defined in any one of the preceding embodiments.

[0403] 43. A cell population, wherein at least 98% of the cells comprised in said population, such as at least 99%, at least 99,5%, at least 99,8% or 100% of the cells comprised in said population, are cells as defined in any one of the preceding embodiments.

[0404] 44. A pharmaceutical composition comprising cells as defined in any one of the preceding embodiments 1 to 40 or a cell population as defined in any one of embodiments 41-44, and a pharmaceutically acceptable excipient.

[0405] 45. A cell according to any one of embodiments 1 to 40 or a cell population according to any one of embodiments 41-44 or a pharmaceutical composition according to embodiment 44 for use as a medical treatment, such as a cell therapy treatment.

[0406] 46. A genetic construct comprising an exogenous sequence as defined in any one of the preceding embodiments, wherein said exogenous sequence is flanked by homology arms.

[0407] 47. A genetic construct comprising an exogenous sequence as defined in any one of the preceding embodiments, wherein said exogenous sequence is flanked by homology arms, wherein said homology arms comprise one or more sequences homologous to the sequence of a targeted insertion site located within an intron of an endogenous gene in a cell genome. 48. A method of manufacturing a cell, such as a cell according to any of the preceding embodiments, or of manufacturing a cell population, such as a cell population comprising cells according to any of the preceding embodiments, wherein said manufacturing method comprises:

[0408] - a gene editing step wherein a cell population is subjected to the targeted insertion of an exogenous sequence or to the targeted insertion of a genetic construct comprising an exogenous sequence flanked by homology arms, wherein said insertion is targeted in an intron of an endogenous gene of interest in the cells of said cell population, wherein said exogenous sequence comprises a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0409] 49. A method of manufacturing according to embodiment 48, wherein said homology arms comprise one or more sequences homologous to the sequence of a targeted insertion site located within an intron of an endogenous gene in the genome of said cells.

[0410] 50. A method of manufacturing according to embodiment 48 or 49, wherein said exogenous sequence comprises: i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0411] 51 . A method of manufacturing of a cell population, wherein said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in embodiments 46 or 47 or an exogenous sequence as defined in any one of embodiments 1 to 45.

[0412] 52. A method according to embodiment 48, wherein upon said gene editing step, the cells in said cell population comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene. 53. A method according to any one of embodiments 48-52, wherein said manufacturing method further comprises: a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest.

[0413] 54. A method of manufacturing a cell population, wherein said manufacturing method comprises:

[0414] - a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct, wherein said genetic construct comprises an exogenous sequence flanked by homology arms, and said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence; and

[0415] - a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous gene of interest.

[0416] 55. A method according to any one of embodiments 48-54, wherein said manufacturing method comprises:

[0417] (a) a gene editing step wherein a cell population is subjected to the targeted insertion of an exogenous sequence or a genetic construct as defined in any one of the preceding embodiments; and

[0418] (b) an enrichment step wherein the cells in said cell population are sorted based on the detection of the presence and / or absence of the protein encoded by said endogenous of interest.

[0419] 56. A method according to embodiment 55, comprising

[0420] (b) an enrichment step wherein the cells in said cell population are sorted based on the absence of the protein encoded by said endogenous of interest.

[0421] 57. A method according to any one of embodiments 48-56, wherein said manufacturing method comprises: (a) a gene editing step wherein a cell population is subjected to targeted insertion of an exogenous sequence or a genetic construct as defined in any one of the preceding embodiments; and

[0422] (b) an enrichment step comprising:

[0423] (b) (i) a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest; and

[0424] (b) (ii) a sorting step wherein cells in which no presence of the protein encoded by said endogenous of interest is detected are selected, and / or cells in which presence of the protein encoded by said endogenous of interest is detected are deselected, wherein said detection (b)(i) and sorting (b)(ii) steps may be performed successively or concomitantly.

[0425] 58. A method according to any one of embodiments 48-57, wherein upon said gene editing step, the cells in said cell population comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene.

[0426] 59. A method according to any one of embodiments 48-58, wherein an enriched cell population is obtained upon said enrichment step, and at least 50% of the cells in said enriched cell population, such as at least 52%, 54%, 56%, 58%, 60%, 64%, 68%, 70%, 75%, 78% or 80%, 85% or 90% of the cells in said enriched cell population, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene.

[0427] 60. A method according to any one of embodiments 48-59, wherein an enriched cell population is obtained upon said enrichment step, and at least 93% of the cells in said enriched cell population, such as at least 94%, 95%, 96%, 97% or 98% of the cells in said enriched cell population, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene, i.e. comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest.

[0428] 61 . A method according to embodiment 60, wherein upon said gene editing step, at least 98% of the cells in said enriched cell population, preferably at least 99%, at least 99,5%, at least 99,8%, at least 99,9% and even more preferably 100% thereof, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene, i.e. comprise the biallelic insertion of said exogenous sequence within said intron of said endogenous gene of interest. A method according to any one of embodiments 48-61 , wherein said at least 50% cells, such as at least 52%, 54%, 56%, 58%, 60%, 64%, 68%, 70%, 75%, 78% or 80%, 85% or 90%, cells in said enriched cell population are cells as defined any one of embodiments 1 to 40. A method according to any one of embodiments 48-62 wherein said exogenous sequence comprises:

[0429] - a first sequence (shutdown sequence) comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and

[0430] - a second sequence (transgene sequence) comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally

[0431] - one or more further sequences (transgene sequences), each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence. A method according to any one of embodiments 48-63 wherein said exogenous sequence comprises:

[0432] - a first sequence (shutdown sequence) comprising a splice acceptor sequence, a stop codon and a polyA sequence, and a coding sequence located after said splice acceptor sequence and before said stop codon. A method according to any one of embodiments 48-64, wherein said transgene coding sequence(s) is selected from the list consisting of

[0433] CD55 gene, encoding CD55 protein wild type or an analogue thereof, PDL1 gene, encoding PDL1 protein wild type or an analogue thereof, CD47 gene, encoding CD47 protein wild type or an analogue thereof, HLA-E gene, encoding HLA-E protein, wild type or an analogue thereof, HLA-F gene, encoding HLA-F protein wild type or an analogue thereof; a B2M-HLA fusion gene, encoding a B2M-HLA fusion protein, such as a B2M-HLA-E and / or a B2M-HLA-G fusion protein(s), such as B2M / HLA-E*0101 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein, optionally with a (G4S)4 linker and a signal peptide, or B2M / HLA-E*0103 nucleic acid sequence encoding a B2M / HLA-E*0101 fusion protein, optionally with a (G4S)4 linker and a signal peptide; a suicide switch gene encoding a suicide switch protein. A method according to embodiment 63 or 64, wherein

[0434] - said first sequence (shutdown sequence) comprising a splice acceptor, exon 3 of B2M gene, a 3’UTR sequence and 4 repeats of SV40 polyA sequence, and

[0435] - said one or more further sequences (transgene sequences) comprise a CD47 transgene coding sequence encode gene and further comprise a B2M-HLA-E and / or B2M-HLA-G transgene coding sequence. A method according to any one of embodiments 48 to 66, wherein the endogenous gene of interest is selected from the list consisting of B2M (p2 microglobulin) gene, CIITA (class II, major histocompatibility complex transactivator) gene, TRAC (T-cell receptor alpha constant) gene, HLA-A gene, HLA-B gene, HLA-C gene, CD155 (also known as poliovarius receptor or PVR) gene, CD122 (also known as interleukine-2 receptor subunit beta or IL2RB) gene, B7-H3 (B3 homolog 3, also known as cluster of differentiation 276 or CD276) gene and TCR-gene. A method according to embodiment 67, wherein the endogenous gene of interest is B2M endogenous gene. A method according to any one of embodiments 48-68, wherein said exogenous sequence comprises a HLA-G or HLA-E transgene coding sequence, wild type or an analogue thereof. A method according to any one of embodiments 48-69, wherein said endogenous gene is the B2M (p2 microglobulin) gene, and / or said exogenous sequence comprises a HLA-G or HLA-E transgene coding sequence, wild type or an analogue thereof, and / or a CD55 transgene coding sequence, wild type or an analogue thereof. A cell or a method according to any one of the preceding embodiments, wherein said exogenous sequence comprises a shutdown sequence comprising a splice acceptor, exon 3 of B2M gene, a 3’UTR sequence and 4 repeats of SV40 polyA sequence, and comprises one or more transgene sequences selected from transgene sequence UCOE- CAG promoter-CD47 gene and transgene sequence EF1a promoter -HLA-E gene. A cell or a method according to any one of the preceding embodiments, wherein said exogenous sequence comprises a shutdown sequence comprising -sA-b2mEx3_3UTR- sv40pAx4 and a transgene sequence comprising UCOE-CAG-CD47 and a transgene sequence comprising -EF1a-HLAE sequences. A cell or a method according to any one of the preceding embodiments, wherein said exogenous sequence comprises a shutdown sequence comprising -sA-b2mEx3_3UTR- sv40pAx4 and a transgene sequence comprising UCOE-CAG-CD47-EF1 a-HLAE sequence. 74. A cell or a method according to any one of the preceding embodiments, wherein said exogenous sequence comprises sequence SEQ ID NO 33.

[0436] 75. A method according to any one of embodiments 48-74 wherein said cells are human cells, such as a genetically engineered human cells.

[0437] 76. A method according to any one of embodiments 48-75 wherein said cells are selected from any one of the groups consisting of:

[0438] - a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC);

[0439] - a cell derived from a stem cell and which may be at any differentiation stage therefrom;

[0440] - a mature cell;

[0441] - a cell derived from a pluripotent stem cell, such as any cell type derived from a pluripotent stem cell;

[0442] - a differentiated cell obtained by culturing a pluripotent cell under differentiation conditions, such as in presence of differentiation factors;

[0443] - a non-natural cell, such as a differentiated cell obtained by genome engineering of a pluripotent cell;

[0444] - a beta cell, an INS+ and NKX6.1+ double positive cell or a C-peptide+ / NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+ / NKX2.2+ double positive cell;

[0445] - a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell;

[0446] - an exosome cell;

[0447] - an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell;

[0448] - a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a cardiomyocyte, a retinal cell, a retinal pigment epithelium cell;

[0449] - a cortical neural progenitor cell;

[0450] - a mesenchymal stem cell;

[0451] - an hematopoietic stem cell (HSC);

[0452] - multipotent stem cells such as hematopoietic stem cells (HSC); and

[0453] - differentiated hematopoietic cells such as T cells, macrophages, NK cells.

[0454] 77. A cell population obtained by a method of manufacturing according to any one of embodiments 48-76. 78. A cell obtained by a method of manufacturing according to any one of embodiments 48- 76.

[0455] 79. An enriched cell population obtained by a method of manufacturing according to any one of embodiments 48-77 wherein at least 50% of the cells in said enriched cell population, such as at least 52%, 54%, 56%, 58%, 60%, 64%, 68%, 70%, 75%, 78% or 80%, 85% or 90% of the cells in said enriched cell population, comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene.

[0456] 80. An enriched cell population obtained by a method of manufacturing according to any one of embodiments 48-77 wherein at least 50% of the cells in said enriched cell population, such as at least 52%, 54%, 56%, 58%, 60%, 64%, 68%, 70%, 75%, 78% or 80%, 85% or 90% of the cells in said enriched cell population, are cells as defined in any one embodiments 1 to 40 and 60-76.

[0457] 81 . A cell according to any one of embodiments 1-40 and 71-74 or a cell population according to any one of embodiments 41-43 and 77-80 for use as a medical treatment.

[0458] 82. A cell or a cell population for use according to embodiment81 , wherein said medical treatment is a cell therapy.

[0459] 83. A cell or a cell population for use according to embodiment 82, wherein said cell therapy is cell replacement therapy.

[0460] 84. A cell or a cell population for use according to anyone of embodiments 81-83, for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.

[0461] 85. A cell or a cell population for use according to anyone of embodiments 81-84, for use in the treatment, the cure, or the prevention of a chronic disease selected from the group consisting of diabetes, type 1 diabetes, type 2 diabetes, dry macular degeneration, retinitis pigmentosa, neurological disease, Parkinson’s disease, heart disease, tissue fibrosis, cirrhosis, hearing loss, corneal blindness, stroke, chronic heart failure, chronic kidney disease or cancer.

[0462] 86. A cell or a cell population for use according to anyone of embodiments 81-84, for use in the treatment, the cure, or the prevention of an acute disease selected from the group consisting of bacterial lung infections, such as ventilator acquired bacterial pneumonia or hospital acquired bacterial pneumonia.

[0463] 87. A cell or a cell population for use according to embodiment 82, wherein said cell therapy is CAR-T cell therapy. 88. A cell or a cell population for use according to anyone of embodiments 81-84, for use in the treatment, the cure, or the prevention of a cancer disease.

[0464] 82. A cell or a cell population for use according to embodiment 81 , wherein said medical treatment is gene therapy.

[0465] Embodiments A1-A15 are further contemplated herein.

[0466] Embodiment A1 : A cell comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises

[0467] • a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0468] Embodiment A2: A cell according to embodiment A1 , wherein said exogenous sequence comprises

[0469] • a splice acceptor (SA) sequence, a stop codon such as a Tristop sequences and preferably two or more stop codons such as two successive Tristop sequences, a coding sequence and two or more successive polyA sequences, such as three or more, and preferably four or more polyA sequences, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequence(s).

[0470] Embodiment A3: A cell according to embodiment A1 or A2, wherein said exogenous sequence comprises

[0471] • a splice acceptor (SA) sequence, two successive triple stop sequences, a coding sequence which comprises exon 3 of B2M gene or a portion thereof, four successive polyA sequences, such as polyA sequence derived from SV40, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequences.

[0472] Embodiment A4: A cell according to embodiment A1 comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0473] Embodiment A5: A cell according to embodiment A4 wherein said first sequence comprises a splice acceptor sequence, a coding sequence, a 3’UTR sequence or a portion thereof, a stop codon and a polyA sequence.

[0474] Embodiment A6: A cell population comprising cells as defined in any one of the preceding embodiments A1 to A5.

[0475] Embodiment A7: A cell population according to embodiment A6, wherein at least 93% such as at least 94%, 95%, 96%, 97% or 98% of the cells comprised in said population are cells as defined in any one of the preceding embodiments.

[0476] Embodiment A8: A cell population, wherein at least 98% of the cells comprised in said population, such as at least 99%, at least 99,5%, at least 99,8% or 100% of the cells comprised in said population, are cells as defined in any one of the preceding embodiments. Embodiment A9: A pharmaceutical composition comprising cells as defined in any one of the preceding embodiments A1 to A5 or a cell population as defined in any one of embodiments A6 to A8, and a pharmaceutically acceptable excipient.

[0477] Embodiment A10: A cell according to any one of embodiments A1 to A5 or a cell population according to any one of embodiments A6 to A8 or a pharmaceutical composition according to embodiment A9 for use as a medical treatment, such as a cell therapy treatment.

[0478] Embodiment A1 1 : A a genetic construct comprising said exogenous sequence flanked by homology arms, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

[0479] Embodiment A12: A method of manufacturing a cell population, wherein said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in any one of the preceding embodiments. Embodiment A13: A method according to embodiment A12, wherein said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in embodiment A11 ; and a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest.

[0480] Embodiment A14: A method according to embodiment A13, wherein said manufacturing method comprises:

[0481] (a) a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in any one of the preceding embodiments; and

[0482] (b) an enrichment step comprising:

[0483] (b) (i) a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest; and

[0484] (b) (ii) a sorting step wherein cells in which no presence of the protein encoded by said endogenous of interest is detected are selected, and / or cells in which presence of the protein encoded by said endogenous of interest is detected are deselected, wherein said detection (b)(i) and sorting (b)(ii) steps may be performed successively or concomitantly.

[0485] Embodiment A15: A genetic construct comprising an exogenous sequence, wherein said exogenous sequence comprises

[0486] • a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

[0487] Examples

[0488] Example 1. Genetic constructs and exogenous sequences

[0489] Several genetic constructs have been prepared comprising exogenous sequences as listed in Table 5 flanked by homology arms as listed in Table 8.

[0490] In constructs #1 to #5, the exogenous sequence comprises a shutdown sequence (first sequence) and no transgene sequence (second sequence), thus the homology arms are located on 5’ and 3’ of the shutdown sequence. In constructs #6 to #12, the exogenous sequence comprises a shutdown sequence (first sequence) and one or more transgene sequences (second and further sequences), thus homology arms are located on 5’ of the shutdown sequence and on 3’ of the last transgene sequence. Said homology arms are designed to target a location within exon 1 of human B2M gene and to allow insertion of the exogenous sequence therein upon cut by CRISPR MAD7 nuclease, as further explained in Example 2.

[0491] Said genetic constructs have been prepared using well known genetic editing techniques. Table 5A: Exogenous sequences

[0492] Table 5B: Sequence of shutdown sequences of Table A:

[0493] Cells were subjected to transfection with above mentioned genetic constructs and to targeted insertion thereof, as explained in Example 2. Thereafter, the cells were subjected to the detection of HLA-I proteins (HLA-A, HLA-B and HLA-C) on the cell surface membrane, measured by Flow (i.e. Fuorescence activated cell sorting (FACS)), using antibody W6 / 32 after - / + I FNy treatment for 24h: “+IFNy” indicates cells which have been subjected to interferon-gamma (IFNy) treatment, by adding IFNy in the cells culture medium. “-IFNy” indicates cells which have not been subjected to interferon-gamma treatment. I FNy treatment forces expression of B2M gene. As B2M protein is necessary to allow translocation of HLA-I protein on cell surface, absence of detection of HLA-I proteins on a cell surface means absence of expression of B2M protein in the cell and detection of HLA-I proteins means there is expression of B2M protein in the cell.

[0494] The results are reported graphically in Figure 5 (Fig. 5). The results are also reported in Table 6 right end column as percentages of cells positive for HLA-A, HLA-B and / or HLA-C proteins expression / detection on cell membrane surface. Most have been tested in more than one clone.

[0495] Table 6. Overview of Figure 5:

[0496] This shows that:

[0497] - the presence of a non-coding sequence, such as B2M-exon3, in the shutdown sequence improves endogenous gene shutdown efficiency: see #8-10 compared to #6-7, and #3-4 compared to #1 ,2 and 5;

[0498] - the presence of a non-coding sequence, such as B2M-exon3, in the shutdown sequence in presence of one or more transgene sequences, such as two transgene sequences, in the exogenous sequence improves endogenous gene shutdown efficiency: see #8-10 compared to #6-7; and - the presence of the sequence comprising a splice acceptor, such as SA, a non-coding sequence, such as B2M-exon3, a 3’-UTR sequence and one or more polyA “pA” sequences, such as 2 pA derived from SV40 or 4 pA derived from SV40, or 4 synthetic pA derived from SV40, in the shutdown sequence in presence of one of more transgene sequences in the exogenous sequence improves endogenous gene shutdown efficiency: see #8-10. Example 2: Genetic construct and insertion thereof in B2M intron 1

[0499] A genetic construct has been prepared according to the illustration in Fig.1 .C and Fig .1 .D (with SA) and in Fig. 7. The construct was designed to target insertion within intron 1 of B2M driven by MAD7 nuclease system. The sequence of the selected target site is shown in Table 7 along with the sequence of the guide RNA used to guide the nuclease to the targeted insertion site and the sequences of the homology arms flanking the genetic construct. Table 7: Nucleotide sequences used in Example 1

[0500] Example 3: Shutdown efficiency in beta-like cell E1 C3 stem cells were subjected to transfection with above mentioned genetic constructs #4 or #11 as defined in Table 5A and 5B for targeted insertion thereof, according to standard electroporation and targeted insertion methods.

[0501] As positive control, E1 C3 stem cells were subjected to electroporation with a guideRNA cutting in Exonl of endogenous B2M gene. As a result, the B2M gene is shutdown.

[0502] The genetic modifications of the tested cell lines are summarized in below Table 8.

[0503] Table 8: Genetic modifications to E1C3 in Example 3

[0504] The cells were then differentiated into beta-cells. Several differentiation protocols have been disclosed and may be used in the present case, such as the differentiation protocol described in patent application published as W02024008810. Upon culture in presence of interferon gamma (IFNy) 100ng IFNy / mL of culture medium for 24 hours at 37C, the cells were subjected to the detection of HLA-I proteins (HLA-A, HLA-B and HLA-C) on the cell surface membrane, measured by Flow (i.e. Fluorescence activated cell sorting (FACS)), using anti-HLA-A, -B and -C antibody clone W6 / 32, commercially available from numerous antibody providers, and according to standard FACS protocols well known in the art .

[0505] The results are reported in Figures 7 A and B and Figure 8.

[0506] The mean fluorescence intensity (MFI) on edited cells (biallelic Tl, clone G5_E8 and F5_G9, with sequence #11) is 0,03% or 0,04% of the MFI on WT cells.

[0507] Comparatively, the mean fluorescence intensity (MFI) on edited cells edited with sequence #4 (biallelic Tl, clone 8E9, with sequence #4) is 0,58% of the MFI on WT cells.

[0508] There is a 10 fold difference, suggesting that sequence #11 allows for more robust shutdown efficiency than sequence #4.

[0509] This shows that sequence #11 improves the shutdown efficiency of the targeted endogenous gene compared to sequence #4.

[0510] This shows that an exogenous sequence comprising a shutdown sequence and 1 or 2 transgene sequences improves the shutdown efficiency of the targeted endogenous gene compared to a sequence which comprises only a shutdown sequence.

[0511] Example 4: Shutdown efficiency in beta-like cell

[0512] Fig. 9 shows B2M shutdown efficiency measured by FACS on

[0513] - beta-like cells derived from E1C3 stem cells (rows 1-3 “WT+, WT HLA-A2” and “WT+ HLA_A2”),

[0514] - beta-like cells derived from E1C3 stem cells which have been subjected to guide RNA targeted cutting in Exonl of endogenous B2M gene (“B2M KO”) as positive control with knocked-out B2M gene (rows 4-5 “KO HLA-A2S” and “KO+ HLA-A2"),

[0515] - beta-like cells derived from E1C3 stem cells which have been subjected to the targeted insertion of a genetic construct comprising exogenous sequence #1 1 as listed in Table 5 (rows 6-7 “303 HLA-A2” and “303+ HLA-A2”), with (rows 1 , 3, 5 and 7) or without IFNy treatment, applying methods indicated in Example 3. Fig.10 shows B2M shutdown efficiency measured by TCR dextramer assay on the same cells as tested in Fig.9.

[0516] The TCR dextramer assay was performed on 2D differentiated beta-like cells, either wild type cells (“WT” cells), or subject to exon-targeted B2M knock-out (“B2M KO” cells) or to intron- targeted of exogenous sequence #1 1 ( ”303” cells). The cells were subjected to overnight incubation with IFNy 100 ng / ml in BC06 media (“+”) while a batch of each was not (“-“).

[0517] The cells were then subjected to a high-affinity, PE conjugated TCR dextramer assay with NY-ESO1 peptide loaded.

[0518] The results reported in Fig.10 show that only WT cells with IFNy stimulation bind to TCR dextramer. Intron-targeted ”303” cells bind to TCR dextramer at the same level as exon- targeted B2M knock-out cells.

[0519] These results show that:

[0520] • In the absence of IFNy, wild type (“WT”) E1 C3-derived beta-like cells display only low cell surface levels of HLA class I proteins, while HLA class II and HLA-E proteins are not detectable;

[0521] • In the presence of IFNy, cell surface levels of HLA class I are strongly upregulated on E1 C3 WT-derived beta-like cells (as measured by antibodies targeting HLA-A2, B2M, or HLA-A,B,C), while HLA-E signal shows only a minimal increase;

[0522] • Compared to the tested B2M exon knock-out (rows 4-5), all B2M intron shutdown clones (rows 6-7) display a faint signal for HLA class I.

[0523] • All B2M-edited clones display less than 0,6% of the HLA class I signal detected on WT E1 C3-derived beta-like cells. This rudimentary signal, if at all representing functional HLA class I, can be regarded as unsuitable for effective peptide presentation to T cells.

[0524] Example 5: Enrichment efficiency

[0525] As described in Example 3, E1 C3 stem cells were subjected to transfection according to standard electroporation and targeted insertion methods with genetic constructs comprising:

[0526] • an exogenous sequence comprising a shutdown sequence, a transgene sequence CAG promoter-CD47wt gene and a transgene sequence EF1 a promoter and B2M- HLA-E"wt" gene, for insertion in endogenous B2M intron 1 using guide RNA g367 and a CRISPR nuclease ; and

[0527] • an exogenous sequence comprising a shutdown sequence, a transgene sequence CAG promoter-PD-L1wt gene and a transgene sequence EF1a promoter-CD55wt gene, for insertion in endogenous CIITA intron using guide RNA g403 and a CRISPR nuclease; and

[0528] • an exogenous sequence comprising HSV-TK for integration in CDK1 using guide RNA g440 and Cre recombinase.

[0529] The above has been performed either in absence or in presence of nedisertib, acting as integration enhancer.

[0530] Upon transfection, the cells were subjected to FACS for sorting of HLA-I negative cells and for enrichment of these cells. HLA-I negative cells are cells for which no cell surface HLA-I protein is detected. These are the cells in which successful biallelic targeted integration of the exogenous sequence in targeted B2M endogenous gene intron 1 has occurred.

[0531] The FACS results reported in Fig. 15 and summarized in below Table 9 show the proportion of HLA-I negative cells out of the total cell population obtained after gene editing.

[0532] Table 9: Editing efficiency: Proportion of cells with no HLA-I proteins detected on the cell surface in FACS, out of the total cell population after gene editing:

[0533] The proportion of cells with successful biallelic targeted integration of the exogenous sequence in targeted B2M endogenous gene intron 1 is of about 48% with integration enhancer and lower, of about 3.62% without integration enhancer.

[0534] Upon enrichment, a cell population enriched in HLA-I negative cells is obtained. From this enriched cell population, 53 cells have been picked randomly, cultured into 53 clones, and each of these 53 clones have been subjected to PCR for analyses of the actual gene editing in their respective genomes. The PCR analyses allowed detection of non-integration, mono allelic integration and bi-allelic integration of the exogenous sequence in the targeted intronic locus. The result from this PCR analysis shows that, in all 53 clones, the billalelic targeted integration has successfully occurred in the targeted intronic locus.

[0535] This shows that, one single sorting and enrichment step, using FACS in this instance, has allowed obtaining a cell population which comprises a very high proportion, 100% in this instance, of cells bearing the desired billalelic targeted integration, hence the desired endogenous gene shutdown and the desired transgene(s) integration.

[0536] This also shows that one single sorting and enrichment step allows obtaining a cell population which comprises a very high proportion, of 100% or close to 100%, of cells bearing the desired targeted intronic integration hence the desired shutdown and transgene(s) integration, even if starting from a cell population comprising a low proportion, such as 3.62% or 30.5%-48% in this instance, of said gene edited cells of interest upon the gene editing step. This benefit is made possible using the genetic construct and exogenous sequence as defined herein.

[0537] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS1 . A cell comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises• a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

2. A cell according to claim 1 , wherein said exogenous sequence comprises• a splice acceptor (SA) sequence, two successive triple stop sequences, a coding sequence which comprises exon 3 of B2M gene or a portion thereof, four successive polyA sequences, such as polyA sequence derived from SV40, and optionally a 3’UTR sequence or a portion thereof located after said coding sequence and before said polyA sequences.

3. A cell according to claim 1 comprising an exogenous sequence located within an intron of an endogenous gene of interest and in both alleles thereof, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

4. An exogenous sequence or a genetic construct comprising an exogenous sequence, wherein said exogenous sequence comprises- a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

5. A genetic construct according to claim 4, comprising said exogenous sequence flanked by homology arms, wherein said exogenous sequence comprises i. a first sequence comprising a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon, and ii. a second sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence, and optionally iii. one or more further sequences, each further sequence comprising a promoter, a transgene coding sequence, a stop codon and a polyA sequence.

6. A method of manufacturing a cell population, wherein said manufacturing method comprises: a gene editing step wherein a cell population is subjected to the targeted insertion of an exogenous sequence as defined in claim 4 or a genetic construct as defined in any one of claims 4 to 5 in an intron of an endogenous gene of interest in the cells of said cell population.

7. A method of manufacturing a cell population, wherein said manufacturing method comprises:- a gene editing step wherein a cell population is subjected to the targeted insertion of an exogenous sequence or to the targeted insertion of a genetic construct comprising an exogenous sequence flanked by homology arms, wherein said insertion is targeted in an intron of an endogenous gene of interest in the cells of said cell population, wherein said exogenous sequence comprises a splice acceptor sequence, a stop codon and a polyA sequence, and optionally a coding sequence and / or a 3’UTR sequence or a portion thereof located after said splice acceptor sequence and before said stop codon.

8. A method according to claim 6 or 7, wherein said manufacturing method comprises: a gene editing step wherein a cell population is subjected to targeted insertion of an exogenous sequence or a genetic construct as defined in claim 4 or 5; anda detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest.

9. A method according to any one of claims 6 to 8, wherein said manufacturing method comprises:(a) a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in any one of the preceding claims; and(b) an enrichment step wherein the cells in said cell population are sorted based on the absence of the protein encoded by said endogenous of interest.

10. A method according to any one of claims 7 to 9, wherein said manufacturing method comprises:(a) a gene editing step wherein a cell population is subjected to targeted insertion of a genetic construct as defined in any one of the preceding claims; and(b) an enrichment step comprising:(b) (i) a detection step wherein the cells in said cell population are subjected to the detection of the presence and / or absence of the protein encoded by said endogenous of interest; and(b) (ii) a sorting step wherein cells in which no presence of the protein encoded by said endogenous of interest is detected are selected, and / or cells in which presence of the protein encoded by said endogenous of interest is detected are deselected, wherein said detection (b)(i) and sorting (b)(ii) steps may be performed successively or concomitantly.11 . A method according to any one of claims 7 to 10, wherein said endogenous gene is the B2M ( 2 microglobulin) gene, and / or said exogenous sequence comprises a HLA-G or HLA-E transgene coding sequence, wild type or an analogue thereof, and / or a CD55 transgene coding sequence, wild type or an analogue thereof.

12. A method according to any one of claims 9 to 1 1 , wherein an enriched cell population is obtained upon said enrichment step, and at least 93% of the cells in said enrichedcell population comprise said exogenous sequence inserted within said targeted intron in both alleles of said endogenous gene.

13. A cell population obtained by a method of manufacturing according to any one of claims 6 to 12, wherein at least 93%, such as at least 94%, 95%, 96%, 97% or 98%, of the cells comprised in said population are cells as defined in any one of claims 1 to 3.

14. A pharmaceutical composition comprising cells as defined in any one of claims 1 to 3 or a cell population as defined in claim 13, and a pharmaceutically acceptable excipient.

15. A cell according to any one of claims 1 to 3 or a cell population according to claim 13 or a pharmaceutical composition according to claim 14 for use as a medical treatment, such as a cell therapy treatment.

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