WO2017105350A1 - A method of generating a mammalian stem cell carrying a transgene, a mammalian stem cell generated by the method and pharmaceuticals uses of the mammalian stem cell - Google Patents

Abstract

The present invention relates to a method of generating a mammalian stem cell carrying a transgene, the method comprising inserting a transgene into the genome of the mammalian stem cell by means of zinc finger nuclease (ZFN) mediated integration. The invention also relates to a mammalian cell obtained by this method as well as to a pharmaceutical composition containing such a mammalian stem cell. The invention further relates to a method of treating a patient having a disease, the method comprising administering the patient a mammalian stem cell of the invention. In illustrative embodiments the disease is a disease associated with a deficiency of a gene or deficiency of the expression of the gene such as a gene selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland. The blood coagulation factor may be selected from the group consisting of factor VII, factor VIII and factor IX and the disease may be any form of hemophilia, i.e. hemophilia A, hemophilia B or hemophilia C.

Description

A METHOD OF GENERATING A MAMMALIAN STEM CELL CARRYING A

TRANSGENE, A MAMMALIAN STEM CELL GENERATED BY THE METHOD

AND PHARMACEUTICALS USES OF THE MAMMALIAN STEM CELL

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] The present application claims the right of priority of US provisional application

62/267,056 filed with the US Patent and Trademark Office on 14 December 2015, the entire content of which is incorporated herein for all purposes.

FIELD OF THE INVENTION

[002] The present invention relates to a method of generating a mammalian stem cell carrying a transgene, a mammalian stem cell generated by the method and pharmaceuticals composition and uses of the mammalian stem cells. The invention is also directed to methods of treating a disease or disorder comprising administering a mammalian stem cell or a pharmaceutical composition containing a mammalian stem cell of the invention to a subject in need thereof.

BACKGROUND OF THE INVENTION

[003] The bedrock of haemophilia A treatment is factor VIII (FVIII) protein replacement to restore haemostatic capacity to a level sufficient to enable normal blood coagulation during activities of daily living. Plasma-free recombinant FVIII products should now be the treatment of choice (Grillberger et al, 2009; Franchini & Lippi, 2010). Compared to on-demand treatment, regular prophylaxis with plasma-free recombinant FVIII protein products greatly reduces the frequency of acute bleeding episodes, chronic musculoskeletal disability and improves health- related quality of life (Manco- Johnson et al, 2007; Plug et al, 2008; Walsh & Valentino, 2009; Collins et al, 2010; Gringeri et al, 2011; Manco-Johnson et al, 2013). However, many patients do not receive optimal, or even any, FVIII replacement at all, largely because of the high cost estimated at US$ 138,000 - US$300,000 annually for a young child, depending on body weight, clinical phenotype and individual FVIII pharmacokinetics (Bernstein, 2007; Manco-Johnson et al, 2007; Roosendaal & Lafeber, 2007). Of the current global population of about 140,000 people with haemophilia A, 75% receive little or no FVIII replacement (Mannucci, 2011 ; World Federation of Hemophilia, 2013). Even when FVIII products are affordable in high income countries, regular prophylaxis is associated with frequent breakthrough bleeding (Collins et al, 2009; Walsh & Valentino, 2009), while the need for frequent intravenous access limits acceptance, especially among children for whom effective early intervention is especially important (Ljung, 2007; Santagostino & Mancuso, 2008).

[004] The fact that high quality FVIII replacement products now available are too costly for more than half the world's population of haemophilia A patients motivates attempts to develop alternative therapies. In vivo gene therapy using viral vectors is appealing for monogenic FVIII deficiency. Although it has not yet achieved the same success as gene therapy for haemophilia B (Nathwani et al, 2014), improvements in FVIII transgene expression and packaging in AAV vectors appear promising (High et al, 2014), as are approaches to minimize immune responses to AAV vectors (Masat et al, 2013).

[005] An alternative strategy is non-viral delivery of a FVIII transgene into autologous cells ex vivo. Proof of concept was demonstrated in a clinical trial of autologous dermal fibroblasts transfected ex vivo with a plasmid that delivered a B domain-deleted FVIII transgene expressed from the fibronectin promoter (Roth et al, 2001). FVIII-secreting autologous fibroblasts implanted in four of six patients with severe haemophilia A achieved measurable, albeit modest, therapeutic efficacy for up to 10 months. Since then, several programmable nucleases with the potential to modify genomes with high precision have emerged and can be delivered by non- viral vectors. Among these, zinc finger nuclease (ZFN) technology is currently most advanced towards possible clinical applications. A phase 1 clinical trial of ZFN-mediated CCR5 inactivation in autologous T cells of 12 patients with chronic HIV infection reported no adverse event that could be ascribed to the use of ZFN (Tebas et al, 2014). Nonetheless, there is heightened awareness of potential oncogenic complications because clinical trials of transgene integration mediated by gammaretroviral vectors have been marred by treatment-induced leukaemias (Hacein-Bey-Abina et al, 2003; Hacein-Bey-Abina et al, 2008; Howe et al, 2008; Stein et al, 2010; Avedillo Diez et al, 2011 ; Gaspar et al, 2011). The occurrence of these life- threatening complications has made biosafety of all genome-modifying techniques crucially important. [006] The extent and nature of off-target genome modifications in ZFN-treated cells have not been comprehensively evaluated although various methods have been employed. Specificity of genome modification has been assessed by screening bioinformatically predicted off-target sites (Hockemeyer et al, 2009), in vitro cleavage of biased libraries (Pattanayak et al, 2011) or sequencing the integration sites of integrase-defective lentiviral vectors (Gabriel et al, 2011). The issue of largely non-overlapping off-target sites generated by different methods (Gabriel et al, 2011; Pattanayak et al, 2011) has been only partially resolved by a machine-learning classifier (Sander et al, 2013) and there remain non-trivial method-dependent discrepancies in off-target site identifications. If, as has been suggested, the binding energy of ZFNs across the whole genome is very low (Hendel et al, 2014), poor correlation between in silico predicted and actual off-target sites is not surprising. Furthermore, it is evident that no single technique for interrogating the genome suffices to reveal off-target modifications comprehensively (Hendel et al, 2014; Pattanayak et al, 2014), neither are there consensus standards for assessing biosafety ex vivo currently. Moreover, unintended clinical effects result from the interplay of multiple factors (such as disease context, cell type, selection of target site, nuclease and donor DNA design, ZFN concentration and conditions of nuclease treatment) which are likely to be specific for different treatment contexts. Given such complexity, it would be desirable to have mammalian cells carrying a transgene, for example, a transgene encoding a factor VIII polypeptide or any other polypeptide the deficient expression or production thereof is associated with a disease or disorder that are safe to use, for example, in gene-therapy and in which the transgene has been stably and site-specifically intergrated.

[007] Accordingly, it is an object of the invention to provide such mammalian cells that meet this need. It is thus also an object of the invention to provide methods of generating such mammalian cells.

SUMMARY OF THE INVENTION

[008] This object is accomplished by the methods, cells, and pharmaceutical compositions having the features of the independent claims. [009] In a first aspect, the invention provides a method of generating a mammalian stem cell carrying a transgene, the method comprising inserting a transgene into the genome of the mammalian stem cell by means of zinc finger nuclease (ZFN) mediated integration.

[0010] In a second aspect, the invention provides a mammalian stem cell carrying a transgene obtained by a method of (the first aspect of) the invention.

[0011] In a third aspect, the invention provides a pharmaceutical composition containing a mammalian cell of (the second aspect of) the invention.

[0012] In a fourth aspect, the invention provides a method of treating a method of treating a patient having a disease, the method comprising administering to the patient a mammalian stem cell of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0013] As explained above, in a first aspect the invention is directed to a method of generating a mammalian stem cell carrying a transgene, the method comprising inserting a transgene into the genome of the mammalian stem cell by means of zinc finger nuclease (ZFN) mediated integration.

[0014] In this context, the term "transgene" is used herein in accordance with its regular meaning in the art to describe a segment of a coding nucleic acid such as DNA that contains a gene sequence that has been isolated from one cell type (or organism) and is introduced into a different cell type (or organism). This non-native nucleic acid (DNA) segment as used herein will retain the ability to produce either a desired RNA molecule or a desired polypeptide (protein) in the transgenic cell (or organism). The transgene may also alter the normal function of the genetic code of the obtained transgenic cell. In general, the nucleic acid molecule (DNA sequence) is incorporated into a germ line of the mammalian stem cell used here.

[0015] Any mammalian stem cell can be used in the present invention, for example, embryonic stem cells, adult stem cells or natal stem cells, to mention only a few. The term "stem cell" as used herein refers to any cell that has the capacity to self-renew indefinitely or to be pluripotent or multipotent and to be able to differentiate (spontaneously under suitable conditions) in multiple cell types or tissue types such as, for example, endothelial cells, epithelial cells, fibroblasts, myocytes or neuronsm to mention only a few. The cells may be derived of any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, dog, cat, sheep, monkey, ape, horse, a macaque or a human, with cells of human origin being preferred in one embodiment.

[0016] In illustrative embodiments the stem cell used in the present invention is selected from the group consisting of a stem cell isolated from the amniotic membrane of the umbilical cord (which is interchangeable also referred to herein as "cord lining stem cell"), a stem cell isolated from Wharton's Jelly of the umbilical cord, a stem cell isolated from the amniotic membrane of the placenta, a stem cell isolated from the endothelium or the subendothelial layer of the umbilical cord vein, and a stem cell isolated from umbilical cord blood. In a partiuclar embodiment, the cord lining stem cell is an epithelial or a mesenchymal stem cell that is described in International patent application WO 2006/019357 or the corresponding published US patent application US2006/078993 or issued US patent 9,085,755. Accordingly, such epithelial or mesenchymal stem cells of the amniotic membrane of the umbilical cord can be isolated as described in WO 2006/019357, US patent application 2006/078993 or US patent 9,085,755. Stem cells from Wharton's Jelly of the umbilical cord, stem cell isolated from the amniotic membrane of the placenta, stem cells isolated from the endothelium or the subendothelial layer of the umbilical cord vein, and stem cells isolated from umbilical cord blood are well known to the person skilled in the art, see for example, the references cited in US patent 9,085,755. See in this respect for example, also Stubbendorf et al, Stem Cells and Development, Volume 22, Number 19, 2013, DOI: 10.1089/scd.2013.0043.

[0017] As said above the mammalian stem cell can be of any origin, it may be selected in illustrative embodiments from the group consisting of a human stem cell, a canine stem cell, a feline stem cell, an equine stem cell, a stem cell of an ape, or a stem cell of a macaque.

[0018] In case a human cord lining stem cell, either a mesenchymal or epithelial cord lining stell cell is used in the present invention, the transgene is integrated by means of the method of the present invention into the AAVS 1 locus (present on human chromosome 19 q 13.3-qter) of a human cord lining stem cell. The AAVS 1 locus is well known to the person skilled in the art and have been described, for example, by Kotin, Linden and Berns in the EMBO Journal vol. 11 no. 13 pages 5071 - 5078, 1992. [0019] In one embodiment of the method of the invention, the method comprises inserting the transgene by means of a mutated zinc finger nuclease.

[0020] In this context, it is noted that the term "Zinc finger nuclease" is used with its standard meaning in the art as, for example used by Guo et al, 2010, cited herein and Doyon et al, 2011, cited herein. In short, zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. A zinc finger nuclease is a site-specific endonuclease designed to bind and cleave DNA at specific positions. There are two protein domains. The first domain is the DNA binding domain, which consists of eukaryotic transcription factors and contains the zinc finger protein. The second domain is the nuclease domain, which typically consists of the Fokl restriction enzyme and is responsible for the catalytic cleavage of DNA. The restriction enzyme Fokl (also termed Fokl herein) is naturally found in Flavobacterium okeanokoites and is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. In accordance with this disclosure, any zinc finger nuclease can be used in the present invention. The Fokl (cleavage) domain can be used in combination with any suitable zinc finger protein. The zinc finger nuclease can either be a homo-dimer or a hetero-dimer. The Fokl restriction enzyme can comprise a sequence of SEQ ID NO. 6 or a sequence comprising only the catalytic domain of this sequence. This catalytic domain is depicted in below Table 1 in bold letters in SEQ ID NO. 6. This catalytic domain comprises 196 amino acids.

[0021] In illustrative embodiments the zinc finger nuclease comprises at least one mutation in the Fokl cleavage domain Fokl, to provide a variant with enhanced cleavage activity. For example, the mutations/substitutions as described herein can correspond to the positions of wildtype Fok I sequence of SEQ ID NO. 6.

[0022] In illustrative embodiments, the mutated zinc finger nuclease may comprise E490K and/or I538K substitutions in one or both monomers of the zinc finger nuclease. Additionally or alternatively, the mutated zinc finger nuclease may comprise Q468E and I499L substitutions in one or both monomers of the zinc finger nuclease. Additionally or alternatively, the mutated zinc finger nuclease may comprise Q468E and I499L substitutions in the left monomer and E490K and I538K substitutions in the right monomer of the zinc finger nuclease. Thus, the mutated zinc finger nuclease may comprise a sequence of SEQ ID NOs. 7 and/or 8.

[0023] In further illustrative embodiments, the mutated zinc finger nuclease may additionally or alternatively comprise the S418P and K441E substitutions in both the right and left monomer of the zinc finger nuclease. Thus, the mutated zinc finger nuclease may comprise a sequence of SEQ ID NOs. 9, 12 or 13. This zinc finger nuclease has been described by Guo et al, 2010 and is also referred to as "Sharkey variant".

[0024] In one illustrative embodiment and as described by Guo et al, 2010 the catalytic domain of the Sharkey variant may have the following amino acid (SEQ ID NO: 9) sequence at sequence positions 384 to 579 according to the numbering a used by Guo et al, taking from Supplementary Figure 2 of Guo et al). The corresponding nucleic acid sequence of the Sharkey cleavage domain has been deposited in GenBank with accession number HM 130522:

384 Gin Leu Val Lys Ser Glu 390 Leu Glu Glu Lys Lys Ser Glu Leu Arg His 400 Lys Leu Lys Tyr Val Pro His Glu Tyr He 410 Glu Leu He Glu He Ala Arg Asn Pro Thr 420 Gin Asp Arg He Leu Glu Met Lys Val Met 430 Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg 440 Gly Glu His Leu Gly Gly Ser Arg Lys Pro 450 As2_Gly Ala He Tyr Thr Val Gly Ser Pro 460 He Asp Tyr Gly Val He Val Asp Thr Lxs 470 Ala Tyr Ser Gly Gly Tyr Asn Leu Pro He 480 Gly Gin Ala Asp Glu Met Gin Arg Tyr Val 490 Glu Glu Asn Gin Thr Arg Asn Lys His lie 500 Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro 510 Ser Ser Val Thr Glu Phe Lys Phe Leu Phe 520 Val Ser Gly His Phe Lys Gly Asn Tyr Lys 530 Ala Gin Leu Thr Arg Leu Asn His He Thr 540 Asn Cys Asn Gly Ala Val Leu SerVal Glu 550 Glu Leu Leu He Gly Gly Glu Met He Lys 560 Ala Gly Thr Leu Thr Leu Glu Glu Val Arg 570 Arg Lys Phe Asn Asn Gly Glu He Asn Phe 579 which three-letter amino acid code sequence corresponds to the following one letter amino acid code sequence,

QLVKSELEEKKSELRHKLKYVPHEYIELIE IARNPTQDRILEMKVMEFFMKVYGYRGEHLGGSRKPDGAIYTVG SP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQ LTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INF (SEQ ID NO: 9). [0025] In the above depicted amino acid sequence of SEQ ID NO: 9, the three amino acids Asp 450, Asp 467 and Lys 469 of the catalytic center are highlighted in bold, underlining and italics. Likewise, the mutations Ser418Pro and Lys441Glu are highlighted in bold, underlining and italics.

[0026] In further embodiments of the method of the present invention, the mutated zinc finger nuclease may additionally or alternatively (e.g. additionally to E490K, I538K, Q468E and/or I499L substitutions) comprise the amino acid substitutions S418P, K441E and H537R in the right Fokl monomer and the amino acid substitutions S418P, K441E and N496D in the left Fokl monomer. Thus, the mutated zinc finger nuclease may comprise a sequence of any one of SEQ ID NOs. 8, 9, 10 or 11 or may comprise a sequence having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92%, 93%, 94%, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of SEQ ID NOs. 10, 11, 14 and/or 15. These mutations/substitutions inzinc finger nuclease have been described by Doyon et al, 2011 , cited herein and is also referred to as "Enhanced Sharkey variant". In the above depicted amino acid sequence of SEQ ID NO: 9, the wildtype sequence at positions 496 (Asn496) and 537 (His537) is highlighted in underlining and italics. The mutations His537Arg and Asn496Glu are thus not shown in SEQ ID NO: 9. It is clear to the skilled person that the the mutated zinc finger nuclease may additionally or alternatively comprise the amino acid substitutions Q486E, I499L, E490K, and/or I538K as also described by Doyon et al. as cited elsewhere herein.

[0027] In further embodiments, the mutated zinc finger nuclease (the obligate heterodimer) may comprise the two amino acid substitutions E490K and I538K in the Fokl monomer fused to the right AAVS 1 homology arm and the two amino acid substitutions Q468E (or T486E) and I499L in the monomer fused to the left homology arm. These substitutions (E490K, I538K in the Fokl monomer fused to the right AAVS 1 homology arm and the two amino acid substitutions Q468E (or T486E) and I499L) are also not shown in SEQ ID NO:9 but again the wild-type sequence is highlighted in underlining and italics at these sequence positions. It should be noted here that is this clear to the person skilled in the art that any zink finger nuclease that comprises the above-explained mutations in the respective Fokl monomer (either in the homo- dimeric or a hetero-dimeric zinc finger nuclease) as long as the Fok monomer provides the desired functionality to ingerate the transgene into the genome of the mammalian stem cell, for example, into the AAVS 1 locus (present on human chromosome 19 ql3.3-qter of human cord lining stem cells. Thus, any functional fragment or mutant of the respective Fokl monomer may be used in the method of the present invention. In this context it is further clear to the skilled person that the positions of the mutations in Fokl as described herein all correspond to respective positions in the sequence of wildtype Fokl as e.g. shown in SEQ ID NO. 6. That means that e.g. S418P indicates a S to P mutation at position 418 of wildtype Fokl of SEQ ID NO. 6.

[0028] In accordance with this disclosure, any zinc finger protein can be present and used in a zinc finger nuclease employed herein. For example, the zinc finger nuclease may comprise a zinc finger protein selected from the group of a Cys2His2-like zinc finger protein having the amino acid sequence motif X2-Cys-X2,4-Cys-Xi2-His-X3,4,5-His, where X can be any amino acid, and number indicates the number of residues (SEQ ID NO. 16; see also Table 1 below), a Gag- knuckle zinc finger protein Treble-clef, a zinc ribbon zinc finger protein or a Zn2/Cys6 zinc finger protein, to name only a few illustrative examples. In further illustrative embodiments of the method of the invention the zinc finger protein may be selected from the group consisting of a P3 zinc finger protein, an E2C (E6) zinc finger protein, an E5 zinc finger protein, an E4 zinc finger protein or an E3 zinc finger protein (cf. Guo et al, 2010, supra in this respect).

[0029] The integration into a genome of the mammalian stem cells can be carried out at any any suitable temperature, for example, at 37°C but also at a higher temperature (for example, up to 42°C) or at a lower temperature. In some embodiments of the method of invention the integration reaction is carried out at a temperature range between about 25 °C and about 32°C. In exemplarily embodiments the integration reaction is carried out at a temperature of about 30°C.

[0030] In the method as described herein, the integration of the transgene is typically carried out by transfection. In some embodiments the transfection is carried out using a single plasmid that delivers both monomers of the zinc finger nuclease. In these embodiments that make use of a single plasmid, this single vector may preferably be transfecting employing the Sharkey or Enhanced Sharkey AAVS1 zinc finger nuclease monomers. In accordance with the above disclosure the transfection may be carried out using transient hypothermia. [0031] As mentioned above, any desired transgene can be integreated into a mammalian stem cell as described here. Usually, the transgene may be any nucleic acid that is suitable for gene therapy, meaning that the recombinant expression of the nucleic acid molecule can ameriolate, treat or prevent a disease in a host, typically a mammal including a human that suffers from a (genetic) dysfunction that leads to a deficiency in the production of a peptide such as hormone or any desired polypeptide. In illustrative embodiments, the transgene is selected from the group of gene (nucleic acid molecule) encoding a blood coagulation factor and a gene (nucleic acid molecule) encoding a protein hormone secreted by an endocrine gland. The blood coagulation factor, may, for example, be selected from the group including but not limited to, factor VII, factor VIII and factor IX. For example, Factor VIII may have a sequence of any one of SEQ ID NOs. 1 , 2 and/or 3 or may have a sequence having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92%, 93%, 94%, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of any one of SEQ ID NOs. 1, 2, and/or 3. In other examples, the deficiency may be a deficiency of the expression or secretion of a protein hormone that is secreted by an endocrine gland and that is associated with an endocrine deficiency. Such a deficiency of the protein hormone that is associated with an endocrine deficiency may be selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

[0032] In one embodiment the transgene is a gene (nucleic acid molecule) that encodes a chimeric factor VIII polypeptide. This chimeric factor VIII polypeptide may be a chimeric protein that has human segments or domains and segments or domains from a non-human mammal. In illustrative embodiments, the transgene may encode a chimeric factor VIII polypeptide that comprises human and porcine domains. An illustrative example of such a human-porcine chimeric protein may be a transgene that encodes a chimeric factor VIII polypeptide that comprises or consists of porcine Al and A3 domains, human signal peptide, the human A2 domain, a residual human B domain and human C 1 and C2 domains (cf. Sivalingam et al, 2014). Such a chimeric factor VIII may have a residual human B domain that comprises the first 266 amino acids of the B domain and eight glycosylation sites. As an illustrative example, the transgene may encodes the chimeric factor VIII polypeptide "Hybrid FVIII_KON lab" (SEQ ID NO: 4) having a length of 1709 amino acids. Alternatively, the transgene may encode a polypeptide having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92%, 93%, 94%, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of polypeptide "Hybrid FVIII_KON lab of SEQ ID NO: 4. In another embodiments, the transgene may encode the molecule termed "Hybrid FVIII_Doering lab" herein having a length of 1467 amino acids (SEQ ID NO: 5) that is described in Doerin et al, Molecular Therapy vol. 17 no. 7, 1145-1154 July 2009.

[0033] In this context, it is noted that by "identity" or "sequence identity" as used herein is meant a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present invention means the percentage of pair-wise identical residues - following (homology) alignment of a sequence of a polypeptide of the invention with a sequence in question - with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.

[0034] In accordance with above disclosure, the invention provides a mammalian stem cell carrying a transgene that is obtained by a method as disclosed herein. Accordingly, the mammalian stem may be selected from the group consisting of a stem cell isolated from the amniotic membrane of the umbilical cord (cord lining stem cell), a stem cell isolated from Wharton's Jelly of the umbilical cord, a stem cell isolated from the amniotic membrane of the placenta, a stem cell isolated from the endothelium or the subendothelial layer of the umbilical cord vein, and a stem cell isolated from umbilical cord blood.

[0035] The invention is also directed to the therapeutic use of a mammalian stem cell as provided by the present invention. Any disease can be treated using a mammalian stem cell of the invention as long as by recombinant expression, the transgene can, as described above, ameriolate, treat or prevent a disease in a host, typically a mammal including a human that suffers from a (genetic) dysfunction. The disease may be a disease associated with a deficiency of a gene or deficiency of the expression of the gene. In these embodiments the gene may be selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland. The blood coagulation factor may, for example, selected from the group consisting of factor VII, factor VIII and factor IX. Accordingly, the disease may be hemophilia, for example, hemophilia A, hemophilia B or hemophilia C. Alternatively, the disease may be associated with an endocrine deficiency. Succh a disease may be associated with a deficiency of the expression or secretion of a protein hormone secreted by an endocrine gland. The deficiency of the protein hormone that is associated with an endocrine deficiency may be selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

[0036] In accordance with the above, the invention also provides a method of treating a patient having a disease, the method comprising administering the patient a mammalian stem cell or a pharmaceutical composition containing a stem cell as disclosed herein. The disease can be any disease as described above. For treating the subject, the mammalian stem cells of the invention may be administered in any suitable way, for example, including but not limited to, by implantation or injection. The stem cells may for example, be implanted subcutaneously, for example, directly under the skin, in body fat or the peritoneum.

[0037] As evident from the above, the invention also provides a pharmaceutical composition that contains a mammalian stem cell as described herein. Such a pharmaceutical composition may be adapted for implantation or injection, for example, for subcutaneous implantation.

[0038] The invention will be further illustrated by the attached figures in which

[0039] Fig. 1 shows a comparison of site-specific cleavage activities of ZFN constructs. Fig. 1(a) shows a comparison of AAVS1 ZFN variants and transient hypothermia on cleavage efficiency. Genomic DNA from K562 cells which were coelectroporated with pZDonor (see Fig. 7) and the following AAVS1 ZFN variants: obligate heterodimer (OH), Sharkey or Enhanced Sharkey. The OH (obligate heterodimer) ZFN had two amino acid changes in the Fokl monomer fused to the right AAVS 1 homology arm (E490K and I538K; comprising SEQ ID NO. 7) and in the monomer fused to the left homology arm (Q468E and I499L; comprising SEQ ID NO. 8). The Sharkey variant had additional S418P and K441E substitutions in both right and left monomers (comprising SEQ IDs NOs. 12 and 13, respectively). The Enhanced Sharkey variant had additional amino acid substitutions: H537R in the right Fokl monomer; (comprising SEQ ID NO: 14) and N496D in the left Fokl monomer (comprising SEQ ID NO: 15). The positions of the the indicated mutations correspond to positions of the sequence of wildtype Fokl as e.g. shown in SEQ ID NO. 6. That means that e.g. S418P indicates a S to P mutation at position 418 of wildtype Fokl of SEQ ID NO. 6. The vectors carrying the ZFN variants are also further described in detail in Fig. 7. The different ZFN variants were cultured at either 37 °C or 30 °C. pEGFP in each electroporation served as an index of transfection efficiency. Site-specific cleavage was evaluated by restriction fragment length polymorphism. Results shown are the mean ± SD of triplicate densitometric measurements of the AAVS 1 modified locus expressed as a percentage of the combined unmodified and modified locus. Both Sharkey and Enhanced Sharkey ZFNs at 30 °C induced highest efficiencies of donor DNA integration (44.5 and 47.9%, respectively) assessed by restriction fragment length polymorphism (RFLP).

[0040] Fig. 1(b) shows a graphical representation of panel a data. Different percentages of K562 genomic DNA attaining AAVS 1 -specific integration of the 50-bp donor DNA for each ZFN variant (determined from panel a) (left axis) are shown against comparable efficiencies of pEGFP gene transfer by electroporation (right axis). Data are mean ± SD (n = 3). Integration was significantly greater (P < 0.05) for cells incubated at 30 °C compared to 37 °C for all ZFN variants. At 37 °C, significantly greater integration was achieved with Enhanced Sharkey (P = 0.0024) and Sharkey (P = 0.03) compared to OH. At 30 °C, Enhanced Sharkey and Sharkey had comparable activity and the former was significantly more active than OH (P = 0.02).

[0041] Fig. 2 shows AAVS1 locus-specific integration of different size donor DNAs. Fig. 2(a) ZFN-dependent integration of donor DNA. K562 cells were coelectroporated with pEGFP (reporter for transfection efficiency) and pZDonor with or without AAVS 1 ZFN mRNA (see also Fig. 7).

(Left): Brightfield and fluorescence images of transfected cells. Bar = 100 μπι

(Right): Restriction fragment length polymorphism (RFLP) was used to quantify site-specific integration of pZDonor- AAVS 1 (see also Fig. 7b). Two hundred ng of genomic DNA was extracted from cells 4 days after electroporation of 10 μg pZDonor- AA VS 1 in the absence or presence of AAVS1 ZFN. PCR primers amplified a 1.9-kb region spanning the AAVSI integration site. PCR was performed on genomic DNA from cells 4 days after treatment with pZdonor only; or 4 (Day 4) and 16 days (Day 16) after treatment with pZdonor and AAVS 1 ZFN mRNA provided evidence of site-specific integration of 50-bp donor DNA. Control PCR amplified a 900-bp region of the AAVSl locus. Two bands (1- and 0.9-kb) indicated donor integration, while a single 1.9-kb band indicated no integration.

Fig.2 (b) shows the accuracy of Enhanced Sharkey AAVSl ZFN-mediated integration of pZdonor EGFP (see also Fig. 7b).

Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells coelectroporated with pZDonor EGFP and Enhanced Sharkey ZFN with or without G418 selection. Notably, stably integrated cells were selected by culture in G418 (0.8 mg/ml) for 14 days. The intensity and volume of DNA bands were quantified by Quantity One software (Bio-Rad). Highest integration was achieved from stably transfected cells, since the intensity of the DNA bands tends to be highest in the G418 selected cells.

Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in grey; Enhanced Sharkey AAVS 1 ZFN recognition half-sites are underlined in light grey. Control PCR amplified a 900-bp region of the AAVSl locus. WT K562 denotes untransfected control K562 cells.

(c) Accuracy of Enhanced Sharkey AAVS 1 ZFN-mediated integration of pZDonor Hybrid FVIII (see also Fig. 7b).

Left: PCR amplification of the left and right integration junctions performed on genomic DNA of K562 cells electroporated with pZDonor Hybrid FVIII only or coelectroporated with Enhanced Sharkey ZFN followed by G418 selection. Control PCR amplified a 900-bp region of the AAVSl locus. WT K562 denotes untransfected control K562 cells. White vertical lines in the gel images demarcate lanes that were merged for clarity. Integration was only achieved from stably transfected cells.

Right: DNA sequence chromatogram of left (top) and right (bottom) junctional PCR amplicons. Vector sequences are underlined in grey; Enhanced Sharkey AAVS 1 ZFN recognition half-sites are underlined in light grey. [0042] Fig. 3 shows the AAVS1 locus-specific integration of FVIII donor DNA in CLECs.

[0043] Fig. 3(a) is a schematic of donor DNA integrated in AAVS 1 locus by homology- directed integration (not drawn to scale). FVIII transgene (5.1 kb Hybrid FVIII of SEQ ID NO. 3) was expressed from the human ferritin light chain promoter (hFER L). To eliminate the iron regulation of the ferritin promoter, the 5' UTR of FerL has been replaced by 5'UTR of the chimpanzee elongation factor 1 (EF1) gene (Chipanzee EF1 5'UTR). The insert further comprised a splice acceptor (SA), puromycin cassette (2A and Puromycin), a polyadenylation (poly A) sequence, a strong enhancer of the major immediate early enhancer of the human cytomegalovirus (CMV enhancer), and a bovine growth hormone polyadenylation (BGH poly A). Heavy light grey bars indicate homology arms. Dashed light grey lines indicate AAVS1 locus genomic DNA flanking the homology arms. Arrows indicate primers for integration junction and overlapping PCR to document integration of the complete FVIII transgene DNA. Stably integrated cord-lining epithelial cells were resistant to puromycin (0.5 mg/ml for 7 days) (puro-CLECs). Wt-CLECs were untreated with plasmids.

[0044] Fig. 3(b) shows the accuracy of Enhanced Sharkey AAVS1 ZFN-mediated integration of pSA-2A-Puro Hybrid FVIII (see also Fig. 7c). Integration junction PCR (JPCR) has been performed, which checks for correct donor integration. JPCR showed donor DNA integration in intron 1 of PPP1R12C. Left and right JPCR amplified diagnostic 5' and 3' genome junctions, respectively, created by donor integration using primers specific to donor DNA and adjacent genomic regions beyond the homology arms. Control positive PCR amplified a 900-bp sequence in the AAVS1 locus 2 kb away from the integration site. DNA size markers are 10, 3, and 1 kb. The right panel shows two different long PCRs encompassing the full-length transgene performed with donor- and locus-specific primers. Only puro-CLEC genomic DNA was positive for the predicted 6.9- and 4.2-kb amplicons, which were sequenced to confirm integration of the complete FVIII transgene cDNA. DNA size markers of 10, 5, and 3 kb are indicated. White vertical line in the gel image demarcates lanes that were merged for clarity, (c) JPCR amplicons were sequenced to confirm site-specific left and right integration junctions. Donor-specific sequences are underlined in grey and Enhanced Sharkey AAVS1 ZFN binding sites are underlined in light grey. [0045] Fig.3(d) shows primary human cord-lining epithelial cells that were electroporated with donor DNA plasmid and ZFN plasmid. Control CLECs were transfected with FVIII donor plasmid only (Cl-CLEC) or cotransfected with FVIII donor plasmid and ZFNs without puromycin selection (C2-CLEC). Puro-CLECs were transfected like C2-CLEC and subjected to puromycin selection (0.5 mg/ml for 7 days beginning 4 days after electroporation). Stably integrated cord-lining epithelial cells were resistant to puromycin (0.5 mg/ml for 7 days) (puro- CLECs). Wt-CLECs were untreated with plasmids. FVIII activity was then measured in conditioned media of wt- and puro-CLECs 1 day and 37 days post-electroporation. Most FVIII is secreted from puro-CLECs at 1 day post-electroporation. Secretion of puro-CLECs was higher at day 37 than at day 1 while secretion of FVIII was not detectable in Cl-CLC and C2-CLC at day 37. This indicates that only puro-CLECs provided for continuous FVIII secretion, while the other transfected CLECs did not show such secretion over time. Data are mean ± SEM; n = 3.

[0046] Fig. 3(e) shows copy number of total integrated donor DNA and integration junctions at AAVS 1 locus relative to copy number of a control locus on chromosome 19q 13.42. To measure the copy numbers of integrated donor DNA digital droplet PCR (dPCR) has been performed. This technique seperates the sample into a large number of partitions and then the reaction is carried out in each partition individually. This separation allows for a more reliable collection and sensitive measurement of nucleic acid amounts. In particular, in Fig. 3(e) dPCR of puro-CLEC genomic DNA was performed to determine copies of integrated vector, left integration junction, right integration junction and two control loci in chromosome 19ql3.42. Puro-CLEC refers to puromycin-selected cells with stable integration of FVIII transgene 1 month postelectroporation. Reactions performed without template DNA were negative controls. Only the vectors integrated into puro-CLECs could be detected over time. About 300-600 copies were integrated per μΐ. Furthermore, on-target transgene copy number relative to total (on- and off-target) transgene copy number by quantitative genomic PCR data showed no significant difference between the copy number of integration junction and vector amplicons.Data are mean ± SD; n = 4.

[0047] Fig.3(f) shows quantitative RT-PCR of PPP 1 R 12C transcript levels (exons 4-6) in wt-CLECs and puro-CLECs. RT-PCR detects RNA expression, while quantitative RT-PCR is utilized for quantification of RNA. Data are mean ± SEM; n = 3. Quantitative RT-PCR showed that levels of PPP1R12C mRNA in puro-CLECs were reduced by half compared to wt-CLECs (Fig. 3B). Taken together with copy number data, this was consistent with monoallelic on-target integration of the FVIII transgene in intron 1 of PPP1R12C.

[0048] Fig. 4 shows off-target genomic changes associated with Enhanced Sharkey AAVS 1 ZFN-mediated FVIII transgene integration. Genomic DNAs from wt- and puro-CLECs as described in Fig. 3 were amplified with phi29 polymerase (REPLI-g kit; Qiagen). High confidence indels were investigated by PCR-Sanger sequencing. Indel in general is a molecular biology term for the insertion or the deletion of bases in the DNA. Thus, the term indel describes mutations resulting in an insertion of nucleotides and/or a deletion of nucleotides. Two single- base substitutions were present in this position without either insertion or deletion. 3 indels have been found. (Indel 2 could not be sequenced continuously owing to highly repetitive sequence motifs.) Indel 2 amplicon could not be obtained as a continuous sequence.

Fig.4 (a) depicts DNA sequence of indel 1. Indel 1 was a false-positive indel because sequence data showed only two single -base substitutions.

Fig. 4(b) depicts sequence of indel 3. Indel 3 comprised expansion of two different satellite DNAs (weblogo.berkeley.edu). Each light grey and grey rectangle represents a single copy of the respective satellite DNA. Indel 3 comprised insertions of two different satellite DNAs. The larger insertion ( 1 ,489 bp) was a 15 -fold expansion of satellite DNA while the smaller insertion (186 bp) was a twofold expansion of a different satellite.

Fig. 4(c) shows WGS (whole genome sequencing) analysis suggested four chromosomal rearrangements, all having unbalanced genome copy number. Depicted are putative breakpoints of unbalanced structural variants (SVs) in puro-CLEC from bioinformatic analysis of WGS data. All chromosome positions refer to hgl9.

Fig. 4(d) depicts quantitative PCR. As all breakpoint loci in the four structural variants (SV) were predicted to have unbalanced genome copy number, quantitative PCR has been performed to determine the copy number of each breakpoint locus in genomic DNA of puro-CLECs relative to the same breakpoint locus in wild-type CLECs (wt-CLECs). Relative copy number at each breakpoint locus was expressed as the ratio of normalized CT values of puro- and wt-CLECs. Normalization was necessary because SV1-SV4 breakpoint loci were amplified at different annealing temperatures to achieve specificity of amplification. The CT value of actin locus amplification was used to normalize the CT value of each breakpoint locus in the same experiment Breakpoint 2 locus of SVl could not be amplified. The candidate breakpoint loci of SV4 were 8 bp apart and were amplified as a single locus. The ratios of puro-CLEC to wt-CLEC genome copy numbers of 0.97-1.07 at all candidate breakpoints analyzed were not consistent with a substantive frequency of unbalanced structural alterations (Fig. 4(d)). Based on the absence of abnormal chromosomal junctions by PCR amplification and the absence of abnormal genome copy number at breakpoint loci by experimental validation, it was unlikely that ZFN treatment had induced biologically meaningful chromosomal rearrangements. Thus, Fig. 4(d) shows relative genome copy number at putative breakpoints by quantitative PCR. The mean CT value at each breakpoint locus was normalized to its own actin CT value. The ratio of normalized copy number of puro-CLEC:wt-CLEC at each putative breakpoint is shown for SV2 and SV3. Genome copy number could be quantified at only one putative breakpoint of SVl and SV4. Data are mean ± SD of triplicate reactions.

[0049] Fig. 5 shows gene ontology classification of over- and underexpressed genes in puro-CLECs and quantitative RT-PCR of selected transcripts. RNA-seq of wt-CLECs and puro- CLECs as described in Fig. 3 identified 17,751 transcripts in total, of which only, as shown in Fig 5(a) 57 were overexpressed and as shown in Fig. 5(b) 33 were underexpressed at least twofold in puro-CLECs. FVIII was among the overexpressed transcripts. Pathway analysis showed that 10 dysregulated transcripts mapped to cytokine-cytokine receptor interaction by DAVID analysis28 (Benjamini-corrected P = 0.011) consistent with PPPlR12C's role in inflammation (see also Fig. 14). Although seven dysregulated genes were potential proto- oncogenes in a consolidated catalogue of more than 1,600 oncogenes (http://www.bushmanlab.org/links/genelists), none mapped to any of the canonical cancer pathways in KEGG.

Fig. 5(c) shows that quantitative RT-PCR was performed to verify changes in the levels of PPP1R12C and selected transcripts in puro-CLECs as described in Fig. 3. CLECs electroporated without plasmid DNA and of the same number of population doublings served as controls. Intron-spanning exonic primers were used to amplify the endogenous PPP1R12C transcript (exons 4-6), neighbouring genes within a 1-Mb interval centered on the AAVS 1 integration site (LILRB4, ISOC2, PPP6R1, NAT14, ZNF579, FIZl and RDHl 3), potential interacting partners of PPP1R12C predicted by Gene Network Central™ (http://www.sabiosciences.com) and Human Protein-Protein Interaction Prediction (http://www.compbio.dundee.ac.uk) that were significantly altered by RNA-Seq analysis (DUSP1, DUSP6, CDC6 and DUSP16), and a housekeeping gene, GAPDH. Transcript levels were normalized to GAPDH expression and the fold-change in transcript levels in puro-CLECs was expressed relative to wt-CLECs using the 'delta-delta QT) method' (Livak & Schmittgen, 2001). The graph shows dysregulation of only DUSP6, a PPP1R12C interacting partner, whose expression was 4.2-fold increase in puro- CLECs. DUSP6 negatively regulates ERK1/230 and high expression impairs epithelial- mesenchymal transition and tumorigenicity. Data are mean ± SEM; n = 2 experiments per group and 3 replicates per sample.

[0050] Fig. 6 shows Enhanced Sharkey AAVS1 ZFN activity and FVIII transgene secretion in other primary human cell types.

Fig. 6(a) shows AAVS1 ZFN-mediated FVIII transgene integration in adult human primary cells. PCR of genomic DNA from puromycin-resistant human dermal fibroblasts (Fibroblast FVIII stables) and bone marrow stromal cells (BMSC FVIII stables) co-electroporated with FVIII donor plasmid (pSA-2A-Puro Hybrid FVIII; see Fig. 3 and 7c) and Enhanced Sharkey AAVS1 ZFN (enhanced sharkey oblgate heterodimer ZFNs; see Fig. 3 and 7a) showed the presence of integrated vector (521 bp), left (602 bp) and right (551 bp) integration junctions. Control PCR amplified a 510-bp region of the AAVS1 locus. Minus template lanes show negative control reactions performed without genomic DNA. White vertical line in the gel image demarcates lanes that were merged for clarity.

Fig. 6(b) shows FVIII secretion by ZFN-modified cells. FVIII activity in conditioned media of wild type- and puromycin-resistant human dermal fibroblasts ( 152 ± 14 mU/106 cells/24 hours) and bone marrow stromal cells (253.3 ± 6.4 mU/106 cells/24 hours) 6 weeks postelectroporation. Like puro-CLECs, ZFN-modified primary fibroblasts and bone marrow-derived stromal cells also secreted FVIII. Data are mean ± SEM; n = 3.

[0051] Fig. 7 shows ZFN variant constructs.

Fig.7(a) shows AAVS1 ZFN variant constructs. Three variants were obligate heterodimer (OH) ZFN (modified according to Miller, J.C., et al. (2007) NatBiotechnol 25: 778-785) comprising catalytic seqence of SEQ ID NO. 7 (right homology arm) and catalytic sequence of SEQ ID NO. 8 (left homology arm); Sharkey obligate heterodimer ZFN (obligate heterodimer further modified according to Guo, J, et al. (2010). J Mol Biol 400: 96107) comprising catalytic seqence of SEQ ID NO. 12 (right homology arm) and catalytic sequence of SEQ ID NO. 13 (left homology arm); Enhanced Sharkey obligate heterodimer ZFN (Sharkey obligate heterodimer ZFN variant further modified according to Doyon, Y, et al. (2011) Nat Methods 8: 7479) comprising catalytic seqence of SEQ ID NO. 14 (right homology arm) and catalytic sequence of SEQ ID NO. 15 (left homology arm).

Fig.7(b) shows donor constructs with AAVS 1 homology arms. Three donors were pZDonor (50 bp insert and multiple cloning site); pZDonor EGFP (encoding 3-kb EGFP); pZDonor Hybrid FVIII (encoding 9-kb human-porcine FVIII cDNA of SEQ ID NO. 3).

Fig.7(c) shows donor constructs for gene trap strategy. Donor constructs with AAVS 1 homology arms and a splice acceptor sequence to express a promoterless puromycin resistance gene from the endogenous PPP1R12C promoter following integration at the AAVSl locus were (left) AAVS 1 S A-2A-puro-pA donor (Addgene plasmid #22075) which integrates a 1 -kb puromycin resistance gene at the AAVS 1 locus; (center) AA V-CAGGS -EGFP (Addgene plasmid #22212) which integrates a 4.2kb fragment comprised of puromycin resistance gene and an EGFP reporter gene expressed from CAGGS promoter; (right) pSA-2A-Puro Hybrid FVIII which integrates a 9-kb fragment of SEQ ID NO. 3 comprised a puromycin resistance gene and human-porcine FVIII cDNA expressed from the human ferritin light chain promoter.

[0052] Fig. 8 shows time-course of ZFN transcription and effect of mild hypothermia on ZFN protein levels in transiently electroporated CLECs. Experiments shown in Fig. 8(a) and (b) were performed using two individual ZFN plasmids, which had the enhanced sharkey obligate heterodimer mutations (AAVSl right Sharkey E490K; I538K; S418P; K441E; U537R fokl comprising SEQ ID NO. 14 and pSCB AAVSl left Sharkey Q468E; I499L; S418P; K441E; Ν496Ό fokl comprising SEQ ID NO. 15). Key fragments from these 2 plasmids were used to derive the Enhanced Sharkey obligate heterodimer ZFN construct (see also Figure 7).

Fig. 8(a) shows time course of transcription of ZFN constructs. RT-PCR was performed on total RNA from transfected CLECs at the time points indicated after electroporation with plasmid DNA encoding either the left ZFN (top) or right ZFN (bottom) using homology arm-specific primers. Negative controls were reactions performed without reverse transcription (Minus RT PCR). RT-PCR of γ-actin mRNA was the positive control. Densitometric measurements of ZFN transcript bands were normalized to their respective actin levels and expressed as a percentage of ZFN mRNA transcript levels at 8 hours (indicated against each lane in both gels). RT-PCR showed highest levels of ZFN expression 8-48 hours after CLECs were electroporated with AAVS1 ZFN plasmids.

Fig. 8(b) shows time course of ZFN protein expression. Upper panel: Protein immunoblot of FLAG-tagged ZFN proteins in CLECs transfected with a single plasmid encoding both left and right ZFNs and incubated at either 37°C or 30°C for the indicated number of days showed higher abundance of ZFN proteins when cells were exposed to mild hypothermia. Untransfected CLECs (WT) were negative for expression of FLAG-tagged ZFN protein. Lower panel: β- Actin served as loading control.

[0053] Fig. 9 shows site-specific double-strand DNA cleavage and homology-directed repair in primary human CLECs.

ZFN activity was assessed by the Enzymatic Mutation Detection Assay (CEL-1) assay (reporting DNA break repair by nonhomologous end joining). CEL I, a endonuclease isolated from celery, is a nuclease shown to have a high specificity for mismatches, insertions, and deletions in double-stranded DNA. This enzyme has been purified and can be used and used in a mutation detection assay.

Fig. 9(a) shows a comparison of AAVS1 ZFN monomer delivery constructs. Two individual ZFN plasmids, which had the enhanced sharkey obligate heterodimer mutations (AAVS1 right Sharkey E490K; I538K; S418P; K441E; U537R fokl comprising SEQ ID NO. 14 and pSCB AAVS1 left Sharkey Q468E; I499L;S418P; K441E; Ν496Ό fokl comprising SEQ ID NO. 15) were co-electroporated. Headings with Dual ZFN were performed with Enhanced Sharkey obligate heterodimer ZFN construct (in Figure 7). CLECs electroporated with two separate plasmids encoding left or right AAVS 1 ZFNs (2 single ZFNs) or a single plasmid encoding both left and right AAVS 1 ZFNs (Dual ZFN) were incubated at either 37°C for 3 days (37°C) or 37°C for 1 day followed by 30°C for 2 days (30°C). The genomic region spanning the AAVS1 ZFN target site was amplified and digested with CEL-1 nuclease (+) or left undigested (-). PCR amplicons were resolved by 10% polyacrylamide gel electrophoresis, imaged and quantified using BioRad®Gel Doc 2000 transilluminator and QuantityOne software. PCR amplicon from the Surveyor™ mutation detection kit (Transgenomic) was the positive control for CEL-1 nuclease digest. Estimates of the proportion of modified genomic DNA (ZFN cleaved and repaired with indels) in the bulk treated population based on densitometry are reported below the respective lanes in gel images for each treatment condition. CEL-1 assay results showed significantly higher ZFN activity when right and left Enhanced Sharkey AA VS 1 ZFN monomers were delivered as a single construct (43 ± 1.9 % compared to ZFN monomers delivered as two constructs (35 ± 1.8 %, P = 0.045).

Fig.9(b) shows AAVS 1 ZFN-dependent donor DNA integration. Left: CLECs electroporated with pZDonor alone or with dualAAVSl ZFN plasmid, both in the presence of pEGFP, were evaluated for gene transfer efficiency by fluorescence microscopy (original magnification xlOO). Scalebar = 100 μπι.

Center: These cells were analysed for site specific integration of a pZDonor by integration junction PCR. Integration junction PCR was performed with a vector-specific and a genome- specific primer to amplify a 1-kb region spanning the integration junction. Control genomic PCR amplified a 900-bp region of the AAVS1 locus. Right: RFLP assay was performed by digesting PCR amplicons that spanned the integration site with Hind III followed by 5% polyacrylamide gel electrophoresis. Using a single plasmid that delivered both Enhanced Sharkey AAVS 1 ZFN monomers and transient hypothermia. Under these conditions, integration junction PCR and RFLP analysis showed no donor DNA integration in CLECs electroporated with pZDonor only.

[0054] Fig. 10 shows cellular toxicity and genotoxicity induced by donor DNA or AAVS 1 ZFN.

Fig. 10(a) shows dose titration of donor DNA for genotoxicity. Effects of increased donor DNA dosage on inducing DNA double-strand breaks. Two million CLECs were electroporated with 2 μg pEGFP (reporter gene), a fixed dose of AAVS 1 ZFN (5 μg) and increasing doses of pZDonor as indicated. CLEC WT denotes unmodified cells which were not electroporated. Genotoxicity was evaluated by the percentage of phosphorylated H2AX-positive cells on day 4 post-electroporation. In general, analysis of H2AX expression can be used to detect the genotoxic effect of different toxic substances. What can be seen from Fig. 10(a) is that the higher the amount of the donor the higher is the amount of double strand breaks. *Indicates P < 0.01 compared to Donor (30 μg) EP and # indicates P < 0.01 compared to Donor ( 10 μg) + ZFN and Donor (20 μg) + ZFN. Data are mean ± SEM; n = 3.

Fig. 10(b) shows dose titration of donor DNA for cytotoxicity. Cellular toxicity was evaluated by comparing the decline in percentage of GFP-positive cells on day 4 post-electroporation relative to day 1 after electroporation under the same experimental conditions as (a) above. What can be seen from (b) is that at day 4 less GFP is detected for all donor concentrations than on day 1. This means that cell toxicity was higher on day 4 than on day 1. There is also a tendency that cell toxicity is highest when using a donor concentration of 30μg. Data are presented as percentage GFP-expressing cells on day 4 relative to day 1. * Indicates P < 0.01 compared to Donor (10 μg) + ZFN and # indicates P < 0.01 compared to Donor (20 μg) + ZFN. Data are mean ± SEM; n = 3.

Fig. 10(c) shows dose titration of AAVS 1 ZFN for genotoxicity. Effects of increased ZFN DNA dosage on inducing DNA doublestrand breaks. Two million CLECs were electroporated with 2 μg pEGFP (reporter gene), a fixed dose of pZDonor (10 μg) and increasing doses of AAVS1 ZFN as indicated. Genotoxicity was evaluated by the percentage of phosphorylated H2AXpositive cells on day 4 Post-electroporation. EP only denotes CLECs which were electroporated without any added plasmid construct. Cell toxicity was very similar at different donor concentrations tested. * Indicates P < 0.05 compared to Donor ( 10 μg) EP and # indicates P < 0.05 compared to Donor (10 μg) + ZFN (5 μg), Donor (10 μg) + ZFN (10 μg) and Donor (10 μg) + ZFN (30 μg) Data are mean ± SEM; n = 3.

Fig. 10(d) shows dose titration of AAVS 1 ZFN for cytotoxicity. Cellular toxicity was evaluated by comparing the decline in percentage of GFP-positive cells on day 4 post-electroporation relative to day 1 after electroporation under the same experimental conditions as (c) above. Data are presented as percentage GFP-expressing cells on day 4 relative to day 1. Cell toxicity was detected for all different experimental setups used. Cell toxiciyty was higher at 4 days post- electroporation than 1 day post-elcetroporation. Furthermore, cell toxicity was highest when 20 μg ZFN were used, indicates P < 0.05 compared to Donor (10 μg) EP and # indicates P < 0.01 compared to Donor (10 μg) + ZFN (10 μg). Data are mean ± SEM; n = 3. The overall results as depicted in (a)-(d) thus show that varying doses of Enhanced Sharkey AAVS 1 ZFN and donor DNA were tested to determine conditions that induced least cellular toxicity quantified by cell viability and phosphorylated histone H2AX. The overall results showed that coelectroporation of 5-10 μg Enhanced Sharkey AAVS1 ZFN with 10 μg donor DNA induced least cellular toxicity.

Fig. 10(e) shows dose titration of AAVS1 ZFN for cell proliferation. MTS assay of viable CLECS that were untreated (CLEC WT), that received electroporation only (EP only), received 10 μg pZDonor donor DNA only or pZDonor DNA and increasing doses of ZFNs as indicated, 1 day post-electroporation. The MTS assay is a method for sensitive quantification of viable cells in proliferation and cytotoxicity. The method is based on the reduction of MTS tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media. This conversion is thought to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells. The formazan dye produced by viable cells can be quantified by measuring the absorbance at 490-500 nm. Thus, absorbance at 490 nm was quantified using MRX II 96-well plate reader (Dynex). In consistency with the results of the experiments as described in Fig. 10 (a)-(d) most viable cells were detected in CLEC WT, EP only and Donor only treated cells, while cells electroporated with Donor and ZFN showed less proliferation/viability. Co- electroporation of 5-10 μg Enhanced Sharkey AAVS 1 ZFN with 10 μg donor DNA induced least cellular toxicity, indicates P < 0.01 compared to Donor (10 μg) EP. Data are mean ± SEM; n = 4.

[0055] Fig. 11 shows AAVS1 site-specific integration of 1.3-kb donor DNA in CLECs. Fig. 11(a) is a schematic (not drawn to scale) showing homologous recombination -mediated integration of pAAVS-SA-2A-puro-pA donor (Addgene plasmid #22075) into the AAVS 1 locus. The primer pairs for integration junction PCR were Puro LF; Puro LR (left junction); and Puro RF2; AAVS 1 R (right junction). Primers for long PCR anchored beyond the integration site were Puro LF and AAVS1 R.

Fig. 11(b) shows accurate integration of donor DNA. Left and right integration junction PCR (Left JPCR and Right JPCR) and Long PCR (spanning the integrated transgene) performed on 200 ng genomic DNA (equivalent to 30440 cells) stably integrated puromycin-resistant CLECs co-electroporated with pAAVS-SA-2A-puro-pA donor (1-kb puromycin resistance gene) and AAVSl ZFN. Amplicons of the predicted sizes (Left JPCR amplicon, 1.1 kb; Right JPCR amplicon, 1.6 kb) were evidence of donor DNA integration at the AAVSl locus. Control PCR amplified a 900-bp region of the AA VS 1 locus 2 kb away from the integration site. Long PCR performed with genome-specific primers anchored beyond the integration site yielded a 2kb amplicon in the absence of donor DNA integration and a dominant 3-kb amplicon in donor- integrated cells. Control PCR reactions on unmodified wild-type CLECs showed only the 2-kb amplicon. The identities of integration junction PCR and long PCR products were confirmed by sequencing. White vertical lines demarcate regions of gel images that were merged.

[0056] Fig. 12 shows AAVSl site-specific integration of 4.2-kb donor DNA in CLECs. Fig. 12(a) is a schematic (not drawn to scale) showing homologous recombination-mediated integration of pAAV-CAGGS-EGFP (Addgene plasmid #22212) into the AAVSl locus. For integration junction PCR, one primer was specific to the vector (donor DNA) and the other primer was specific to genomic DNA beyond the integration site (left junction: AAVS1F, Puro LR; right junction: GFP RF, Puro RR). Long PCR amplified two overlapping segments to cover the entire length of donor DNA using outer primers specific to genomic DNA beyond the integration site (Long PCR Left: AAVSl F, CAGGS R; Long PCR Right: GFP F, Puro RR). Fig. 12(b) shows accurate integration of donor DNA. Integration junction PCR (Left JPCR and Right JPCR) and overlapping long PCRs (spanning the integrated transgene) were performed on 200 ng genomic DNA (equivalent to 30440 cells) puromycin-resistant CLECs co-electroporated with pAAV-CAGGS-EGFP (4.2 kb donor DNA consisting of promoterless puromycin resistance gene cDNA and CAGGS promoter-EGFP cDNA) and a plasmid construct encoding both left and right AAVSl ZFNs showed donor DNA integration at the AAVSl locus. Products of left and right integration junction PCR (1 kb and 1.3 kb, respectively) were of the correct predicted size. Control PCR amplified a 900-bp region of the AAVS 1 locus 2 kb away from the integration site. Overlapping long PCR encompassing the integrated transgene amplified products of the predicted sizes (Long PCR Left; 2.3 kb, Long PCR Right; 2.5 kb). No amplicons were generated in parallel PCRs performed on unmodified wild-type CLECs. Fig.l2(c) shows durable transgene expression integrated in AAVS1 locus. Brightfield and fluorescence images of CLECs on day 1 (before puromycin selection) and on days 9, 21 and 33 (after puromycin selection) post-electroporation with pAAV-CAGGS-EGFP show that the AAVS1 locus supports durable EGFP expression of donor DNA. Scale-bar = 100 μπι.

[0057] Fig. 13 shows sensitivity of indel detection by targeted deep sequencing. A commercially synthesized DNA fragment (GenScript, Piscataway, NJ) similar to the AAVS 1 locus sequence except for a 5-bp deletion between the ZFN binding half-sites was spiked into the wild-type AAVS 1 locus amplicon at molar ratios of 1 : 10, 1 : 100, 1 :500 and 1 : 1 ,000 to determine the sensitivity of indel detection. Table and graph show the ratios and percentages of mutant amplicons to wild type amplicons, the number of experimentally retrieved mapped reads, the number and percentage of indels detected in these mapped reads for each spike-in concentration. A highly linear correlation (R2 = 0.999) was observed between the known percentage of indels in the spike-in controls and the percentage of indels experimentally determined by deep sequencing. These data establish a detection sensitivity of 0.1%.

[0058] Fig. 14 shows pathway analysis of 90 dysregulated transcripts in puro-CLECs. DAVID analysis of genes which were over-expressed (n = 57) or under-expressed (n = 33) by = 2-fold in puro-CLECs compared to wild-type CLECs were mapped only to chemokine- chemokine receptor interaction. Pathway analysis showed that 10 dysregulated transcripts mapped to cytokine-cytokine receptor interaction by DAVID analysis (Benjamini -corrected P = 0.011), consistent with PPPlR12C's role in inflammation. Although seven dysregulated genes were potential proto-oncogenes in a consolidated catalogue of more than 1,600 oncogenes (http://www.bushmanlab.org/links/genelists), none mapped to any of the canonical cancer pathways in KEGG.

[0059] Fig. 15 shows a comparison of wt-CLEC and puro-CLEC cell proliferation. An MTS assay, which detects prolfieration of cells, was performed 7 days after wt-CLECs and puro- CLECs were plated at an initial density of 100 cells per well (96-well plate). Proliferation of puro-CLECs was slightly but not significantly reduced, possibly reflecting the combined effects of high DUSP6 expression and PPP1R12C haploinsuffciency, the latter being required for completion of mitosis and cytokinesis. Data are mean absorbance readings ± SD; n = 6 per group. P = 0.303.

[0060] Fig. 16 shows site-specific genome modification in different adult primary human cells. (Top) Transfection of adult primary human cells. Brightfield and fluorescence images of primary human cells 1 day post-electroporation with pmaxGFP (Lonza). Bright field pictures show that cells are viable. Flourescent pictures below show GFP-positive cells. Thus, transfection was successful in all tested adult primary human cells. NF123 and KF1 are dermal fibroblasts; BMSCl and BMSC2 are bone marrow -derived stromal cells; ADSCl and ADSC2 are adipose tissue-derived stromal cells. Scale-bar = 100 μπι.

(Bottom) Site-specific AAVS1 ZFN activity in adult primary human cells. A region spanning the AA VS 1 integration site was amplified from genomic DNA extracted from BMSC 1 , BMSC2, ADSCl, ADSC2, NF123 and KF1 electroporated with pZDonor. CEL-1 nuclease digested (+) or undigested (-) PCR amplicons were resolved by 10% polyacrylamide gel electrophoresis. The CEL- 1 assay report DNA break repair by nonhomologous end joining and thus indirectly detects ZFN activity. Positive integration control was a PCR amplicon from genomic DNA provided in the CompoZr® targeted integration kit (Sigma-Aldrich). Transfection efficiency determined by flow cytometry analysis of GFP-positive cells and genome modification efficiency determined by densitometry of cleaved and uncleaved amplicons are shown. Percent genome modification normalized to 100% transfection efficiency is also shown. ZFN-mediated cleavage at the AAVS1 locus was readily induced in primary human dermal fibroblasts, human bone marrow- and adipose tissue-derived stromal cells with overall effciencies of 20% or higher. Data are mean ± SD; n = 3 per group.

[0061] Fig. 17 shows FVIII transgene secretion by different primary human cell types. Primary human bone marrow-derived stromal cells (BMSCl and BMSC2), human adipose tissue-derived stromal cells (ADSC2) and human cord lining epithelial cells (CLEC) secrete high levels of hybrid FVIII following co-electroporation with pmaxGFP (Lonza) and plasmid DNA encoding hybrid FVIII cDNA expressed from a CMV promoter. WT denotes untransfected control cells. Graph shows the percentage of GFP-positive cells (diamonds; right axis) determined by flow cytometry and FVIII activity (bars; left axis) in overnight conditioned media determined using the Coamatic® Factor VIII kit assay (Chromogenix). Using the same conditions which integrated FVIII transgene in CLECs, primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells could also be induced to integrate and secrete transgenic FVIII. Data are mean ± SEM; n = 3.

[0062] Fig. 18 shows hybrid human-porcine B domain-truncated FVIII cDNA and assembly steps. Factor VIII protein consists of six domains: A1-A2-B-A3-C1-C2. The A domains are homologous to the A domains of the copper-binding protein ceruloplasmin. The C domains belong to the phospholipid-binding discoidin domain family, and the C2 domain mediates membrane binding. Activation of factor VIII to factor Villa is done by cleavage and release of the B domain.

Top: Schematic of B domain-truncated human-porcine FVIII cDNA consisting of porcine Al and A3 domains, human signal peptide, A2, residual B (comprising the first 247 amino acids and six glycosylation sites), CI and C2 domains.

Bottom: Overlap PCR steps used in domain assembly. Al and A3 domains were obtained by RT-PCR of total pig liver RNA based on the reference porcine cDNA sequence (NM_214167.1). Human domains were amplified from complete human FVIII cDNA in pSP64-VIII (American Type Culture Collection.

[0063] Fig. 19 shows targeted deep sequencing of in silico predicted 10 most likely AAVS1 ZFN off-target sites. Top-10 predicted off-target sites for AAVS1 ZFNs (OT1-OT10) were evaluated by targeted deep sequencing of amplicons from genomic DNA from Wt -CLECs and Puro-CLECs. Chromosomal loci of OT1-OT10, the corresponding chromosomal positions, total reads mapped, types of indels experimentally detected and their corresponding percentages are summarized. Indels present only in Puro-CLECs are highlighted in italics. All genome coordinates refer to hgl9. Targeted deep sequencing of the predicted ten most likely off-target sites (OT1-OT10) showed 4- and 1-bp deletions at low frequency (1.36 and 1.37%, respectively) only in OT1, an 8q24.3 intergenic locus. No other indels in OT2-OT10 specific to puro-CLECs were detected. [0064] In the following Table 1 sequences that have been used in the present invention are shown:

Coagulati TRYLRIHPQSWVHQIALRMEVLGCEAQDLY

on factor

VIII

Hybrid GTGTCGTGAAAACTACCCCTAAAAGCCACCGGCGTGGCTAGCATGCAAATAGAGCTCTCCAC FVIII CTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCATCAGGAGATACTACCTGGGCG CAGTGGAACTGTCCTGGGACTACCGGCAAAGTGAACTCCTCCGTGAGCTGCACGTGGACACC AGATTTCCTGCTACAGCGCCAGGAGCTCTTCCGTTGGGCCCGTCAGTCCTGTACAAAAAGAC TGTGTTCGTAGAGTTCACGGATCAACTTTTCAGCGTTGCCAGGCCCAGGCCACCATGGATGG GTCTGCTGGGTCCTACCATCCAGGCTGAGGTTTACGACACGGTGGTCGTTACCCTGAAGAAC ATGGCTTCTCATCCCGTTAGTCTTCACGCTGTCGGCGTCTCCTTCTGGAAATCTTCCGAAGG CGCTGAATATGAGGATCACACCAGCCAAAGGGAGAAGGAAGACGATAAAGTCCTTCCCGGTA AAAGCCAAACCTACGTCTGGCAGGTCCTGAAAGAAAATGGTCCAACAGCCTCTGACCCACCA TGTCTCACCTACTCATACCTGTCTCACGTGGACCTGGTGAAAGACCTGAATTCGGGCCTCAT TGGAGCCCTGCTGGTTTGTAGAGAAGGGAGTCTGACCAGAGAAAGGACCCAGAACCTGCACG AATTTGTACTACTTTTTGCTGTCTTTGATGAAGGGAAAAGTTGGCACTCAGCAAGAAATGAC TCCTGGACACGGGCCATGGATCCCGCACCTGCCAGGGCCCAGCCTGCAATGCACACAGTCAA TGGCTATGTCAACAGGTCTCTGCCAGGTCTGATCGGATGTCATAAGAAATCAGTCTACTGGC ACGTGATTGGAATGGGCACCAGCCCGGAAGTGCACTCCATTTTTCTTGAAGGCCACACGTTT CTCGTGAGGCACCATCGCCAGGCTTCCTTGGAGATCTCGCCACTAACTTTCCTCACTGCTCA GACATTCCTGATGGACCTTGGCCAGTTCCTACTGTTTTGTCATATCTCTTCCCACCACCATG GTGGCATGGAGGCTCACGTCAGAGTAGAAAGCTGCGCCGAGGAGCCCCAGCTGCGGAGGAAA GCTGATGAAGAGGAAGATTATGATGACAATTTGTACGACTCGGACATGGACGTGGTCCGGCT CGATGGTGACGACGTGTCTCCCTTTATCCAAATCCGCTCAGTTGCCAAGAAGCATCCTAAAA CTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCC CCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAA GTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTC AGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATT ATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCG TCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGC CAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGAT CCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGG ACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAA TGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTC ACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTT CCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGCAGTTGTCAG TTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTT TCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCT ATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGG GGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGT GACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAG TAAAAACAATGCCATTGAACCAAGAAGCTTCTCCCAGAATTCAAGACACCCTAGCACTAGGC AAAAGCAATTTAATGCCACCACAATTCCAGAAAATGACATAGAGAAGACTGACCCTTGGTTT GCACACAGAACACCTATGCCTAAAATACAAAATGTCTCCTCTAGTGATTTGTTGATGCTCTT GCGACAGAGTCCTACTCCACATGGGCTATCCTTATCTGATCTCCAAGAAGCCAAATATGAGA CTTTTTCTGATGATCCATCACCTGGAGCAATAGACAGTAATAACAGCCTGTCTGAAATGACA CACTTCAGGCCACAGCTCCATCACAGTGGGGACATGGTATTTACCCCTGAGTCAGGCCTCCA ATTAAGATTAAATGAGAAACTGGGGACAACTGCAGCAACAGAGTTGAAGAAACTTGATTTCA AAGTTTCTAGTACATCAAATAATCTGATTTCAACAATTCCATCAGACAATTTGGCAGCAGGT ACTGATAATACAAGTTCCTTAGGACCCCCAAGTATGCCAGTTCATTATGATAGTCAATTAGA TACCACTCTATTTGGCAAAAAGTCATCTCCCCTTACTGAGTCTGGTGGACCTCTGAGCTTGA GTGAAGAAAATAATGATTCAAAGTTGTTAGAATCAGGTTTAATGAATAGCCAAGAAAGTTCA TGGGGAAAAAATGTATCGTCAACAGAGAGTGGTAGGTTATTTAAAGGGAAAAGAGCTCATGG ACCTGCTTTGTTGACTAAAGATGGGAGGACTGAAAGGCTGTGCTCTCAAAACCCACCAGTCT TGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGAC TATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGA AAATCAGAGCCCCCGCAGCTTTCAGAAGAGAACCCGACACTATTTCATTGCTGCGGTGGAGC AGCTCTGGGATTACGGGATGAGCGAATCCCCCCGGGCGCTAAGAAACAGGGCTCAGAACGGA GAGGTGCCTCGGTTCAAGAAGGTGGTCTTCCGGGAATTTGCTGACGGCTCCTTCACGCAGCC GTCGTACCGCGGGGAACTCAACAAACACTTGGGGCTCTTGGGACCCTACATCAGAGCGGAAG TTGAAGACAACATCATGGTAACTTTCAAAAACCAGGCGTCTCGTCCCTATTCCTTCTACTCG AGCCTTATTTCTTATCCGGATGATCAGGAGCAAGGGGCAGAACCTCGACACAACTTCGTCCA GCCAAATGAAACCAGAACTTACTTTTGGAAAGTGCAGCATCACATGGCACCCACAGAAGACG AGTTTGACTGCAAAGCCTGGGCCTACTTTTCTGATGTTGACCTGGAAAAAGATGTGCACTCA GGCTTGATCGGCCCCCTTCTGATCTGCCGCGCCAACACCCTGAACGCTGCTCACGGTAGACA AGTGACCGTGCAAGAATTTGCTCTGTTTTTCACTATTTTTGATGAGACAAAGAGCTGGTACT TCACTGAAAATGTGGAAAGGAACTGCCGGGCCCCCTGCCACCTGCAGATGGAGGACCCCACT CTGAAAGAAAACTATCGCTTCCATGCAATCAATGGCTATGTGATGGATACACTCCCTGGCTT AGTAATGGCTCAGAATCAAAGGATCCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAATA TCCATTCGATTCATTTTAGCGGACACGTGTTCAGTGTACGGAAAAAGGAGGAGTATAAAATG GCCGTGTACAATCTCTATCCGGGTGTCTTTGAGACAGTGGAAATGCTACCGTCCAAAGTTGG AATTTGGCGAATAGAATGCCTGATTGGCGAGCACCTGCAAGCTGGGATGAGCACGACTTTCC TGGTGTACAGCAAGAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTT CAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTC CGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGG CACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTAC ATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAA TTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATA TTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGC AGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAAT GGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTG CCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCT CAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGG AGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCT CCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTT CAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCG CTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGG GCTGCGAGGCACAGGACCTCTACTGAGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATC AGCCT

Hybrid MQIELSTCFFLCLLRFCFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPLGP FVIII_K SVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTWVTLKNMASHPVSLHAVGVS ON lab FWKS SEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLSHVDLVK 1709 As DLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAMDPAPARAQ

PAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHS IFLEGHTFLVRHHRQASLEISP LTFLTAQTFLMDLGQFLLFCHI SSHHHGGMEAHVRVESCAEEPQLRRKADEEEDYDDNLYDS DMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLI IFKNQASRPYNIYP HGITDVRPLYSRRLPKGVKHLKDFP ILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGV QLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMV YEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCDKNTGDYYEDSYED ISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEND IEKTDPWFAHRTPMPKIQNVS S SDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVF TPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPV HYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLF KGKRAHGPALLTKDGRTERLCSQNPPVLKRHQRE ITRTTLQSDQEE IDYDDT ISVEMKKEDF DIYDEDENQSPRSFQKRTRHYF IAAVEQLWDYGMSESPRALRNRAQNGEVPRFKKWFREFA DGSFTQP SYRGELNKHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAE PRHNFVQPNETRTYFWKVQHHMAPTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTL NAAHGRQVTVQEFALFFTIFDETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYV MDTLPGLVMAQNQRIRWYLLSMGSNENIHS IHFSGHVFSVRKKEEYKMAVYNLYPGVFETVE MLPSKVGIWRIECLIGEHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGS INAWSTKEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQF I IMYSLDGKK WQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPP I IARYIRLHPTHYS IRSTLRMELMGCDLNS CSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ KTMKVTGVTTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSL DPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

Hybrid MQLELSTCVFLCLLPLGFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPLGP FVIII_Do SVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPTIQAEVYDTWVTLKNMASHPVSLHAVGVS ering lab FWKS SEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLSHVDLVK

DLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAMDPAPARAQ PAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHS IFLEGHTFLVRHHRQASLEISP LTFLTAQTFLMDLGQFLLFCHI SSHHHGGMEAHVRVESCAEEPQLRRKADEEEDYDDNLYDS DMDVVRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLI IFKNQASRPYNIYP HGITDVRPLYSRRLPKGVKHLKDFP ILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGV QLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMV YEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCDKNTGDYYEDSYED ISAYLLSKNNAIEPRSFAQNSRPPSASAPKPPVLRRHQRD ISLPTFQPEEDKMDYDDIFSTE TKGEDFD IYGEDENQDPRSFQKRTRHYF IAAVEQLWDYGMSESPRALRNRAQNGEVPRFKKV VFREFADGSFTQP SYRGELNKHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYPDD QEQGAEPRHNFVQPNETRTYFWKVQHHMAPTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLI CRANTLNAAHGRQVTVQEFALFFTIFDETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFH AINGYVMDTLPGLVMAQNQRIRWYLLSMGSNENIHS IHFSGHVFSVRKKEEYKMAVYNLYPG VFETVEMLPSKVGIWRIECLIGEHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQY GQWAPKLARLHYSGS INAWSTKEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQF I IMY SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPP I IARYIRLHPTHYS IRSTLRMELM GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEW LQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFT PWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

Wildtype MVSKIRTFGWVQNPGKFENLKRWQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVAIMNQH Fok l DLIYTYKELVGTGTS IRSEAPCDAI IQATIADQGNKKGYIDNWSSDGFLRWAHALGFIEYIN

KSDSFVITDVGLAYSKSADGSAIEKEILIEAISSYPPAIRILTLLEDGQHLTKFDLGKNLGF SGESGFTSLPEGILLDTLANAMPKDKGE IRNNWEGSSDKYARMIGGWLDKLGLVKQGKKEF I IPTLGKPDNKEFI SHAFKITGEGLKVLRRAKGSTKFTRVPKRVYWEMLATNLTDKEYVRTRR ALILEILIKAGSLKIEQIQDNLKKLGFDEVIETIENDIKGLINTGIFIEIKGRFYQLKDHIL QFVIPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMK VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRN KHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMI KAGTLTLEEVRRKFNNGEINF

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGG right SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVKENQTRNKHINPNEWWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm RKFNNGE IN

(catalytic

domain)

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGG left SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMKRYVEENQTRNKHLNPNEWWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm RKFNNGE INF

(catalytic

domain)

Sharkey QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGgHLGG

(catalytic SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKV domain) YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR

RKFNNGE INF

Enhanced QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGgHLGG sharkey SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKV

Fokl right YPSSVTEFKFLFVSGHFKGNYKAQLTRLNRITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR monomer RKFNNGE INF

(catalytic

domain)

Enhanced QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRG HLGG sharkey SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRDKHINPNEWWKV left YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR Fokl RKFNNGE INF

monomer

(catalytic

domain)

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGBHLGG right SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVKENQTRNKHINPNEWWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm + RKFNNGE IN

S418P,

K441E

(catalytic

domain)

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNFTQDRILEMKVMEFFMKVYGYRG HLGG left SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMERYVEENQTRNKHLNPNEWWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm + RKFNNGE INF

S418P,

K441E

(catalytic

domain)

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRG.SHLGG right SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVKENQTRNKHINPNEWWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNRKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm + RKFNNGE INF

S418P,

K441E

and

H537R

(catalytic

domain)

OH-ZFN- QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRG.SHLGG left SRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQAD EMSRY VE E NQ T RD KHLNP NE WWKV homology YPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR arm + RKFNNGE INF

S418P,

K441E

and

N496D

(catalytic

domain)

zinc X2-Cys-X₂,4-Cys-Xi2-His-X₃,4,5-His

finger this three letter code corresponds to one letter code:

protein X2-C-X2,4-C-Xl2-H-X3,4,5-H

(amino

acid where X can be any amino acid, and number indicates the number of residues sequence

motif)

Control 1 cctgccttaaacccagccag

forward

Control 1 atgacct catgct cttggccct cgta

reverse

Control 2 t cccctcccagaaagacctgc

forward

Control 2 t cccctcccagaaagacctgc

reverse

probe tacctaacgcact cctgggtga

Left ttcgggt cacct ct cact cc

genome

specific

forward

Left gacgcgcgtgaggaagagtt c

vector

specific

reverse

Left aggcgcaccgtgggcttgt

probe

Right ctgtggaatgtgtgtcagttag

vector

specific

forward

Right ggct ccat cgtaagcaaacc

genome

specific

reverse

Right cgcct ctgcct ctgagctat

probe

vector cgagatgaccgagtacaag

specific

PCR forward

2 vector gctcgtagaaggggaggttg

9 specific

reverse

3 probe tcaccgagctgcaagaact

0

3 left ZFN ATCCTGTCCCTA

1 half-site

3 right ZFN ACCCCACAGTGG

2 half-site

Table 1 : Sequences used in the present application. The catalytic domain of Fokl is depicted in bold letters in SEQ ID NO. 6. Highlighted in orange are mutations of OH-ZFN (E490K and I538K or Q468E and I499L); highlighted in red are sharkey mutations S418P, and K441E and highlighted in blue are mutations of enhanced sharkey mutants N496D and H537R.

[0065] The invention will also be further illustrated by the following non-limiting Experimental Examples.

[0066] Experimental Examples

[0067] The unmet need for affordable and accessible FVIII replacement motivated the inventors to develop autologous cell therapy using ZFN-mediated FVIII transgene integration in the AAVS 1 locus of primary human cells. This locus is considered, but has not been rigorously shown, to be a genomic safe harbour (Smith et al, 2008; DeKelver et al, 2010; Rio et al, 2014) chiefly because endogenous insulator elements (Ogata et al, 2003) could favour durable transgene expression in different cell types without transactivating neighbouring genes (Smith et al, 2008; Hockemeyer et al, 2009; DeKelver et al, 2010; Zou et al, 2011 ; Coluccio et al, 2013). By combining bioinformatically guided and unbiased interrogations of the genome and transcriptome using a suite of different techniques, it is show herein that AAVS l ZFN with molecular features favouring accurate integration and enhanced nuclease activity integrated a FVIII transgene and induced durable FVIII secretion by primary human umbilical cord-lining epithelial stem cells (CLECs) with negligible off-target effects.

[0068] Results

[0069] Fig 1 shows all plasmid constructs used in the study.

[0070] Evaluation of AAVSl ZFN constructs in K562 cells [0071] Three AA VS 1 ZFN constructs were tested: obligate heterodimer (OH); Sharkey and Enhanced Sharkey (described in Materials and Methods) to quantify integration of pZDonor (50- bp) in K562 cells. We further compared each construct under conditions of mild hypothermia (30°C) (Doyon et al, 2010) and at 37°C. Both Sharkey and Enhanced Sharkey ZFNs at 30°C induced highest efficiencies of donor DNA integration (44.5% and 47.9%, respectively) assessed by restriction fragment length polymorphism (RFLP) (Fig 1). Enhanced Sharkey ZFN was used at 30°C in all subsequent experiments. Under these conditions, integration of 50-bp, 3.75-kb and 9.1-kb donor DNAs into the AAVSl locus of K562 cells was demonstrated by RFLP and integration junction PCR. Sequencing of integration junction PCR amplicons confirmed precise integration of donor DNA into intron 1 of PPP1R12C (Fig 3B, C). Cells electroporated with donor DNA alone in the absence of ZFNs did not show evidence of transgene integration by integration junction PCR and RFLP (Fig 3 A, C). The 9.1-kb donor DNA delivered a hybrid human -porcine B domain-deleted FVIII cDNA (Sivalingam et al, 2014) (Fig 18; see Materials and Methods).

[0072] AAVSl ZFN activity in CLECs (epithelial cord lining stem cells)

[0073] The epithelial cord lining stem cells were isolated as described in WO 2006/019357. RT-PCR showed highest levels of ZFN expression 8-48 hours after CLECs were electroporated with AAVSl ZFN plasmids (Fig 8 A). Two factors were investigated for their effects on ZFN activity assessed by the CEL-1 assay (reporting DNA break repair by non-homologous end joining), integration junction PCR and RFLP (evidence of site-specific donor integration). CEL- 1 assay results showed significantly higher ZFN activity when right and left AAVSl ZFN monomers were delivered as a single construct (43% ± 1.9%) compared to ZFN monomers delivered as two constructs (35% ± 1.8%) (P = 0.045) (Fig 9A). ZFN protein expression was also higher in CLECs subjected to transient mild hypothermia after transfection (37°C for 1 day followed by 30°C for 2 days) (Fig 8B). Thus, subsequent experiments were conducted using a single plasmid that delivered both Enhanced Sharkey AA VS 1 ZFN monomers and transient hypothermia. Under these conditions, integration junction PCR and RFLP analysis showed no donor DNA integration in CLECs electroporated with pZDonor only (Fig 9B).

[0074] Varying doses of AAVS 1 ZFN and donor DNA were tested to determine conditions that induced least cellular toxicity quantified by cell viability and phosphorylated histone H2AX. The overall results showed that co-electroporation of 5 - 10 μg AAVS 1 ZFN with 10 μg donor

DNA induced least cellular toxicity (Fig 10).

[0075] Gene trap integration of FVIII transgene

[0076] As the size of FVIII transgene donor DNA was relatively large (9 kb) and because accurate integration was a paramount goal, we first assessed the accuracy of genome targeting in CLECs using gene trap donor vectors, AAVS1 SA-2A-puromycin-pA and AAV-CAGGS-EGFP (Hockemeyer et al, 2009), which delivered donor DNAs of smaller sizes. PCR of puromycin- resistant CLECs demonstrated integration of 1-kb and 4.2-kb donors, respectively, using genome-specific primers which overlapped the intended integration site (Figs 1 1 and 12). Sequencing of PCR amplicons confirmed complete integration of the 1-kb donor. Most of the integrated 4.2-kb donor was amplified and sequenced (except for a 1-kb GC-rich region within the CAGGS promoter), and showed no insertions, deletions or rearrangements. PCR of integration junctions and donor DNA were negative in untreated CLECs.

[0077] Using the same gene trap strategy, integration junction and long PCR of CLECs co- electroporated with the FVIII transgene donor DNA and Enhanced Sharkey AAVS1 ZFN showed integration of the complete donor in intron 1 of PPP1R12C (Fig 3 A, B). AAVS 1 site- specific PCR and sequencing confirmed insertion of the complete FVIII transgene. Resistance to puromycin selected CLECs which had stably integrated the FVIII transgene (puro-CLECs). FVIII secretion by these cells 37 days post-electroporation was 2, 131 ± 17 mU/10⁶ cells/24 h compared with 952 ± 8 mU/10⁶ cells/24 h 1 day post-electroporation (Fig 3D).

[0078] Quantifying on- and off-target FVIII transgene integration

[0079] Quantifying on-target versus off-target transgene integrations by digital droplet PCR of integration junction amplicons (on-target) versus vector-specific amplicons (on- and off-target integrations) showed no significant difference in copy number of total vector (300 ± 61 copies/μΐ) compared with left junction (312 ± 38 copies/μΐ; P = 0.835) and right junction (360 ± 30 copies/μΐ; P = 0.337) amplicons, suggesting minimal off-target integrations (Fig 3E). RT- PCR showed 2-fold lower PPP1R12C expression in puro-CLECs compared to untreated CLECs, consistent with monoallelic transgene integration (Fig 3F).

[0080] Analysis of in silico predicted off-target sites

[0081] Targeted deep sequencing of the predicted 10 most likely off-target sites (OT1 - OT10) (Hockemeyer et al, 2009) showed 4- and 1-bp deletions at low frequency (1.36% and 1.37%, respectively) only in OT1, an 8q24.3 intergenic locus. No other indels in OT2 - OT10 specific to puro-CLECs were detected (Figure 19). Sensitivity of indel detection was determined by spiking wild-type AAVS1 amplicon with a synthetic amplicon having a 5 -bp deletion at the AAVS1 target site at molar ratios of 1: 10 to 1 : 1000 (mutant: wild type). Deep sequencing detected the spiked deletion at all ratios with a highly linear relationship between spike-in and observed levels (R² = 0.999), establishing a detection sensitivity of 0.1% (Fig 13).

[0082] Whole-genome sequencing

[0083] VarScan analysis (Koboldt et al, 2012) of whole-genome sequencing (WGS) data generated 2736 high-confidence indels specific to puro-CLECs, but none were in silico predicted off-target sites (Cradick et al, 2011; Reyon et al, 2011; Fine et al, 2014). We considered only loci harbouring multiple indels to be true off-target events (Gupta et al, 2011). Genomic coordinates of high-confidence indels revealed 196 loci with 2 - 5 indels each. All were in repetitive DNA regions. Three off-target loci had the highest combined number of indels and SNPs (4 - 6) and also higher proportions (33-50 %) of mapped reads than on-target indels (19- 26%). On-target indels were identified by aligning all reads to a 980-base sequence of the reference AAVS1 locus (hgl9). The ZFN cleavage site was between positions 401 - 430 of this sequence. Four of 21 mapped reads (19%) showed a single A deletion at position 400 of this sequence. Twenty-three of 50 mapped reads (46%) showed either a single T deletion or a single C insertion at position 407. The putative off-target events mapped to intergenic regions in chr2_237939600 (indel 1); chr21_15213700 (indel 2); and chr8_1365500 (indel 3). (Genome coordinates refer to hgl9.) All lacked potential AAVS 1 ZFN-binding sites. The nearest protein- coding genes were COPS8 (54 kb from indel 1), C21orfl5 (1.7 kb from indel 2) and DLGAP2 (84 kb from indel 3). Sequence data of indel 1 showed it to be a false positive indel because it consisted only of two single-base substitutions without an insertion or deletion (Fig 4A). Indel 3 comprised insertions of two different satellite DNAs. The larger insertion (1489 bp) was a 15- fold expansion of satellite DNA while the smaller insertion (186 bp) was a 2-fold expansion of a different satellite (Fig 4B). (Indel 2 could not be sequenced continuously owing to highly repetitive sequence motifs.) Tandem repeats are intrinsically unstable because of replication slippage and unequal sister chromatid exchange during mitosis (George & Alani, 2012). Off- target cleavage by Fokl possibly facilitated repeat expansion by strand invasion (Buard & Jeffreys, 1997). Our data indicate that off -target events caused by Fokl dimerization independent of ZFN binding are rare.

[0084] WGS analysis suggested the presence of four chromosomal rearrangements, all having unbalanced genome copy number. Three were interchromosomal and one was an intrachromosomal structural variant (Fig 4C). Genomic PCR repeatedly failed to detect all putative abnormal chromosomal junctions, suggesting that the rearrangements were another false positive finding. However, as it was important to determine if ZFN treatment had induced structural changes in chromosomes, we employed a different validation method based on relative genome copy number analysis. As all breakpoint loci in the four structural variants were predicted to have unbalanced genome copy number, we used quantitative PCR to determine the copy number of each breakpoint locus in genomic DNA of puro-CLECs relative to the same breakpoint locus in wild- type CLECs (wt-CLECs). Relative copy number at each breakpoint locus was expressed as the ratio of normalized Or values of puro- and wt-CLECs. Normalization was necessary because SV1-SV4 breakpoint loci were amplified at different annealing temperatures to achieve specificity of amplification. The Or value of actin locus amplification, determined in the same experiment, was used to normalize the C_T value of each breakpoint locus. The ratio of puro-CLEC to wt-CLEC C_T values at each breakpoint was calculated as [puro-CLEC breakpoint locus Cr/actin C_T divided by wt-CLEC breakpoint Cr/actin C_T] (Fig 4D). Breakpoint 2 locus of SV1 could not be amplified. The candidate breakpoint loci of SV4 were 8 bp apart and were amplified as a single locus. The ratios of puro-CLEC to wt-CLEC genome copy number of 0.97- 1.07 at all candidate breakpoints analysed were not consistent with a substantial frequency of structural alterations having unbalanced copy number (Fig 4D). Based on the absence of abnormal chromosomal junctions by PCR amplification and the absence of abnormal genome copy number at breakpoint loci by experimental validation, it was unlikely that ZFN treatment had induced biologically meaningful chromosomal rearrangements.

[0085] RNA-seq

[0086] RNA-seq of wt-CLECs and puro-CLECs identified 17,751 transcripts in total, of which only 57 (0.3%) were over-expressed and 33 (0.2%) were under-expressed at least 2-fold in puro-CLECs (Fig 5). FVIII was among the over-expressed transcripts. Pathway analysis showed that 10 dysregulated transcripts mapped to cytokine-cytokine receptor interaction by DAVID analysis (Huang et al, 2009) (Benjamini-corrected P = 0.011) (Fig 14) consistent with PPPlR12C's proposed role in inflammation (Bannert et al, 2003). Although seven dysregulated genes were potential proto-oncogenes in a consolidated catalogue of more than 1,600 oncogenes (http://www.bushmanlab.org/links/genelists), none mapped to any of the canonical cancer pathways in KEGG (http://www.kegg.jp/kegg- bin/show_pathway?map=hsa05200&show_description=show).

[0087] In view of AAVS1 ZFN's minimal footprint on the transcriptome, we broadened our analysis to investigate possible indirect effects of haploinsufficient PPP1R12C expression on its interacting protein partners and downstream effectors. RNA-seq data of 74 other protein phosphatases, 50 myosin-related and downstream genes, 29 known protein interacting partners and 43 neighbouring genes of PPP1R12C within 1 Mb centered on the AAVS 1 integration site showed dysregulation of only DUSP6, a PPP1R12C interacting partner, whose expression was 5.5-fold higher in puro-CLECs. Quantitative RT-PCR confirmed 4.2-fold increase in DUSP6 expression in puro-CLECs (Fig 5). DUSP6 negatively regulates ERK1/2 (Zhang et al, 2010) and high expression impairs epithelial-mesenchymal transition and tumorigenicity (Wong et al, 2012). Proliferation of puro-CLECs was slightly but not significantly reduced (Fig 15), possibly reflecting the combined effects of high DUSP6 expression and PPP1R12C haploinsufficiency, the latter being required for completion of mitosis and cytokinesis (Banko et al, 2011).

[0088] ZFN-mediated cleavage at the AAVS 1 locus was also readily induced in primary human dermal fibroblasts, human bone marrow- and adipose tissue-derived stromal cells with overall efficiencies of 20% or higher (Fig 16). Using the same conditions which integrated FVIII transgene in CLECs, primary human dermal fibroblasts, bone marrow- and adipose tissue- derived stromal cells could also be induced to integrate and secrete transgenic FVIII (Fig 16).

[0089] Discussion

[0090] Recent successes of gene therapy for familial lipoprotein lipase deficiency and haemophilia B (Buning, 2013; Nathwani et al, 2014) have done much to advance the idea, first mooted about fifty years ago (Tatum, 1966), that gene replacement can be both helpful and clinically feasible for some diseases. However, initial forecasts of the timeline for clinical adoption were, in retrospect, sanguine (Friedman, 1992). Oncogenic complications of gene therapy clinical trials in the past decade have made safety the most critical clinical standard, particularly for diseases for which there already are non-genetic treatments and are not urgently life-limiting, such as cancers. Gammaretroviral vectors have long been a favoured workhorse for integrating therapeutic transgenes. Insertional oncogenesis was perceived initially to be largely a theoretical risk because early studies showed no adverse complications in non-human primates and a small number of human subjects (Anderson, 1992). The occurrence of leukaemias and pre- leukaemia in gammaretroviral gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) (Hacein-Bey-Abina et al, 2003; Hacein-Bey-Abina et al, 2008; Howe et al, 2008), chronic granulomatous disease (Stein et al, 2010) and Wiskott-Aldrich syndrome (Avedillo Diez et al, 2011) dispelled this perception and has sharply refocused emphasis on biosafety. Analysis of leukemic cells revealed gammaretroviral vector integrations in the proximity of several oncogenes (LM02, BMI1, CCND2, PRDM16, SETBP1 and MDS- EVI1 ), causing oncogene overexpression. However, precise leukaemogenic mechanisms in these trials remain ill-defined as the same vector used in SCID-X1 patients did not induce leukaemia in a clinical trial of adenosine deaminase deficiency (Cassani et al, 2009), suggesting that the disease context itself sets the threshold for oncogenic transformation which requires dysregulation of more than a single gene (Modlich et al, 2005; Dave et al, 2009). Nonvirally delivered programmable nucleases are being explored as a parallel strategy for achieving safe and efficacious therapeutic transgene integration. In theory, genome editing techniques are well suited for this purpose as it should be feasible to design constructs which avoid integration in potentially hazardous regions i.e. regulatory elements, transcription start sites and within transcription units. There is currently more pre-clinical research experience with ZFN technology than with transcription activator-like effector nucleases (TALENs) and RNA-guided clustered regularly interspaced short palindromic repeat CRISPR/Cas9 systems. Indeed, five clinical trials of ZFN for HIV infection have been completed or are in progress (www.clinicaltrials.gov; accessed on 8 June 2015). A completed trial of 12 HIV-infected patients did not report adverse complications attributable to ZFN up to 36 weeks after a single infusion of autologous T-cells which had been transduced ex vivo with CC7?5-specific ZFNs. Given that oncogenic effects may only appear over a much longer period, subjects in this trial will be observed for 10 years (Tebas et al, 2014).

[0091] Reliable assessment of the genotoxic risks of ZFN and other genome modifying methods that would be considered sufficiently robust for making clinical decisions is a vexing challenge. There is currently no consensus on how modified genomes should be examined and analyzed for unintended potentially genotoxic off-target alterations. Moreover, each application of genome editing must be assessed independently as many technical and biological factors affect the accuracy of genome editing (Porteus & Baltimore, 2003; Miller et al, 2007; Beumer et al, 2008; Pruett-Miller et al, 2008; Doyon et al, 2010; Guo et al, 2010; Jantz & Berg, 2010; Doyon et al, 2011 ; Ramirez et al, 2012; Wang et al, 2012). Furthermore, epigenetic states may account for cell line-dependent variations in cleavage efficiencies (Handel etal, 2009; Lombardo et al, 2011).

[0092] In the present invention, a FVIII transgene was integrated in primary human stem cells (somatic cells) as a realistic model for exploring the feasibility of autologous FVIII cell therapy. Non-haematopoietic primary human cells have limited replicative capacity in vitro which makes derivation and in vitro expansion to scale up single cell clones for clinical therapy impractical. Thus, bulk cell populations of gene-modified CD34⁺ haematopoietic stem cells were used in clinical trials of SCID-X1 (Hacein-Bey-Abina et al, 2014), chronic granulomatous disease (Ott et al, 2006) and Wiskott-Aldrich syndrome (Hacein-Bey-Abina et al, 2015). Rigorously assessing off-target events in bulk cell populations presents a challenge that must be addressed in moving towards clinical cell therapy. It was reasoned here that profiling ZFN- modified CLECs by several different molecular methods would more likely identify induced oncogenic events than reliance on a single technique of characterization. To this end, ZFN- modified CLECs were profiled by targeted deep sequencing of the in silico predicted ten most likely off-target sites and complemented this with unbiased whole genome sequencing. This showed a low frequency (<1.5%) of 4- and 1-bp deletions in only one predicted off-target site. These off-target microdeletions, in an intergenic region of 8q24.3, were 38.2 kb distant from TRAPPC9 (on the 5' side) and 14.6 kb from CHRACl (on the 3' side). TRAPPC9 is thought to be involved in NF-κΒ signalling (Mochida et al, 2009) and CHRACl encodes a histone-fold DNA-binding protein (Poot et al, 2000). The expression of neither gene was dysregulated in puro-CLECs.

[0093] Whole-genome sequencing is used increasingly for unbiased identification of single nucleotide polymorphisms (SNPs), indels and rearranged chromosomes. WGS data obtained at 24x and 28.4x depths of coverage have identified indels (Shigemizu et al, 2013; Ghoneim et al, 2014). However, whereas WGS data at a mean coverage depth of 14x detected SNPs with 95% sensitivity (Meynert et al, 2014), the depth of coverage needed for comparable sensitivity of indel detection is unknown. A further constraint is that analytical methods for inferring indels from WGA data are not as well developed as algorithms for SNPs (Albers et al, 2011). Our results showing a low incidence of indels in ZFN-modified CLECs mirrors a recent report that no SNPs and indels were induced by ZFN correction of SOD1 mutation in human induced pluripotent stem cells (Kiskinis et al, 2014). Although WGS may seem to be ideal for unbiased off-target evaluation, at least two considerations, aside from cost, currently limit its practical utility as a standalone method. Even at high depths of coverage, sequencing artifacts and various bioinformatic filters applied to WGS data can be expected to generate false results. Furthermore, to identify off-target events present at frequencies of 10%, 1% and 0.1% with 95% sensitivity is estimated to require sequencing 15, 150 or 1,500 diploid single-cell clones, respectively (Tsai & Joung, 2014). Detecting a mutation present in 1% of cells sequenced at 500x depth would, on average, be based on only 2.5 reads (Brash, 2015). These considerations make WGS impractical as the sole or primary technique for biosafety assessment given the current state of technology and costs

[0094] Recognizing these limitations, genomic profiling was broadened with quantitative analysis of integration junction and transgene copy number. Two lines of evidence indicated accurate ZFN-mediated FVIII transgene integration in the AAVS1 locus of puro-CLECs. First, on-target transgene copy number relative to total (on- and off-target) transgene copy number by quantitative genomic PCR data showed no significant difference between the copy number of integration junction and vector amplicons (Fig. 3B). This was evidence that very few, if any, integrations were off-target. Second, quantitative RT-PCR showed that levels of PPP1R12C mRNA in puro-CLECs were reduced by half compared to wt-CLECs (Fig. 3F). Taken together with copy number data, this was consistent with monoallelic on-target integration of the FVIII transgene in intron 1 of PPP1R12C.

[0095] RNA-seq generates agnostic whole transcriptome data with greater sensitivity and dynamic range than gene arrays. Given the limitations of genomic analysis alone, it was reasoned here that RNA-seq would provide a complementary whole-genome functional readout that would signal if CLECs had sustained significant ZFN-induced off-target hits within genes and regulatory elements. Although the present study did not profile the expression of non-coding RNAs (ncRNAs), off-target events could also have altered short and long ncRNAs. However, as a major function of ncRNAs is to regulate transcription (Morris, 2011 ; Patil et al, 2014), consequences of altered ncRNA expression would be reflected also in RNA-seq data. In the event, only 0.5% of 17,751 expressed transcripts were dysregulated by 2-fold or more in puro- CLECs compared to wt-CLECs (Fig. 5). It was noted that the dysregulated genes mapped only to the cytokine-cytokine receptor interaction pathway. This was not unexpected because PPP1R12C is a known binding partner of pro-IL16 whose cleavage products engage in T cell immune responses (Bannert et al, 2003; Cruickshank & Little, 2008). None of the dysregulated genes mapped to any of the other 15 canonical cancer pathways in KEGG, although seven were potential proto-oncogenes.

[0096] PPP1R12C encodes a regulatory subunit of protein phosphatase 1, PPl, which is involved in a wide range of important biological processes including mitotic exit, apoptosis, DNA damage response, signalling and metabolism (Hofman et al, 2000; Brady & Saltiel, 2001 ; Bennett, 2005; Kuntziger etal, 2011; Wurzenberger & Gerlich, 2011; Meadows, 2013; Korrodi- Gregorio et al, 2014). PPP1R12C interacts with several proteins including CAMKK1, CDC42BPB, MPRIP, MYL2, MYL5, MYL7, MYL9, MYL10, MYL12A, MYL12B, MYLPF, PHLPP2 (string-db.org), PRKG1 (Surks et al, 1999), pro-IL-16 (Bannert et al, 2003) and SRF (Mulder et al, 2005). Its interacting partners function in cell proliferation and differentiation, cell survival, cell migration, apoptosis, transcription, cytoskeletal organization and signal transduction (nitric oxide-cyclic GMP, Aktl, protein kinases C and A). Given the many potentially critical functions of PPP1R12C and PPl, RNA-seq data of puro-CLECs gave very little evidence that haploinsufficiency of PPP1R12C was functionally deleterious. This was consistent with the tolerance of embryonic stem cells and induced pluripotent stem cells for AAVS 1 ZFN-induced biallelic disruption of PPP1R12C (Smith et al, 2008; Hockemeyer et al, 2009; DeKelver et al, 2010). As the FVIII donor DNA construct in this study was designed not to integrate an exogenous enhancer, the observations made here provide more support for the safe harbour status of the AAVS1 locus.

[0097] ZFNs have been used intentionally to re-create cancer-associated chromosomal translocations (Piganeau et al, 2013). As chromosomal rearrangements could also result from unintended off-target activity, we analysed puro-CLEC WGS data for possible indications of chromosomal structural variants. Four unbalanced chromosomal rearrangements appeared possible. All putative breakpoints were intronic, intergenic or in the 3' UTR (Fig 4C). However, experimental validation by quantitative PCR did not show unbalanced copy number at any of the possible breakpoints examined (Fig. 4D) and predicted fusion transcripts were not detected, suggesting that these were likely false positive results of data analysis.

[0098] Recognising that fresh umbilical cords are the source of CLECs but that autologous cell therapy should be developed for patients of all ages with haemophilia A, three types of readily procured primary human somatic cells were tested too, i.e. dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. All three cell types were permissive for reasonably high levels of DNA cleavage activity using the same AA VS 1 ZFN construct that integrated FVIII transgene in CLECs. Like puro-CLECs, ZFN-modified primary fibroblasts and bone marrow-derived stromal cells also secreted FVIII (Fig 17). This points to potentially wide application of the approach for autologous cell therapy also for adult patients using fibroblasts, bone marrow- and adipose tissue-derived stromal cells whose proficiency for synthesizing and secreting therapeutic proteins is well known (Naffakh et al, 1995; Falqui et al, 1999; Bartholomew et al, 2001 ; Schwenter et al, 2003; Rehman et al, 2004; Kyriakou et al, 2006; Suga et al, 2009; Kakeda et al, 2011).

[0099] In summary, the present study has shown that AAVS1 ZFN designed for high biosafety has broad applicability for autologous cell therapy using several primary human cell types and could be developed as potential FVIII-secreting bioimplants with a low risk of unintended oncogenic effects.

[00100] Materials and Methods

[00101] Cell culture

[00102] K562 cells were purchased from the American Type Culture Collection. Primary human cells (dermal fibroblasts, adipose-derived stromal cells, bone marrow-derived stromal cells and cord-lining epithelial cells) were derived and provided by CellResearch Corporation, Singapore with National University Health System IRB approval. The epithelial cord lining stem cells were isolated as described in WO 2006/019357.

[00103] K562 cells were cultured in Iscove's modified Eagle medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (Hyclone). All primary human cells, except cord- lining epithelial cells, were cultured in Dulbecco's modified Eagle medium (DMEM)-25 mM glucose (Sigma-Aldrich) supplemented with 10% foetal bovine serum. Cord-lining epithelial cells were cultured in Medium 171 (Cascade Biologicals) supplemented with 50 ng/ml IGF- 1 , 50 ng/ml PDGF-BB, 5 ng/ml TGF-βΙ and 5 ng/ml insulin (all from R&D Systems). [00104] AAVS1 ZFNs

[00105] DNA encoding the wild-type catalytic domain of Fokl (pST1374; Addgene) was mutagenized for heterodimerization (Miller et al, 2007). The OH (obligate heterodimer) ZFN had two amino acid changes in the Fokl monomer fused to the right AAVS1 homology arm (E490K and I538K; SEQ ID NO. 7) and in the monomer fused to the left homology arm (Q468E and I499L) (SEQ ID NO. 8). The further variants of OH ZFN were made to enhance cleavage activity:

a) The Sharkey variant had S418P and K441 E substitutions in both right and left monomers (Guo et al, 2010) (SEQ ID NO: 12 and 13).

b) The Enhanced Sharkey variant had additional amino acid substitutions: S418P, K441E and H537R (right Fokl monomer; (SEQ ID NO: 14) and S418P, K441E and N496D (left Fokl monomer) (Doyon et al, 2011) (SEQ ID NO: 15).

Notably, the positions of the the indicated mutations correspond to positions of the sequence of wildtype Fokl as e.g. shown in SEQ ID NO. 6. That means that e.g. S418P indicates a S to P mutation at position 418 of wildtype Fokl of SEQ ID NO. 6.

The final ZFN constructs combined both right and left AAVSl-Fofcl expression cassettes in single plasmids (Fig 7A).

[001] Codon-optimized DNA encoding a pair of zinc finger peptides for the AAVS 1 locus

(Hockemeyer et al, 2009) was commercially synthesized (DNA2.0, USA) and ligated to their corresponding mutagenized Fokl monomers in separate plasmid constructs.

[002] Donor DNA

[003] Three plasmids having a neomycin resistance marker were used to integrate donor

DNAs of increasing sizes into intron 1 of PPP1R12C (Fig 7B):

[004] pZDonor (contains 1500-bp homology to the AAVS1 locus bisected by a 50-bp multiple cloning site; Sigma- Aldrich)

[005] pZDonor EGFP (3.75-kb CMV-promoter-GFP excised from pEGFP-C 1 (Clontech) cloned in pZDonor)

[006] pZDonor Hybrid FVIII (9.1 -kb donor encoding human ferritin light chain promoter- hybrid FVIII cDNA cloned in pZDonor; described below). [007] For integration in primary human cells, donor vectors with a promoterless puromycin resistance selection gene were assembled on pAAVSl SA-2A-puromycin-pA (Addgene plasmid #22075) and pAAV-CAGGS-EGFP (Addgene plasmid #22212) (Fig 7C).

[008] Hybrid FVIII cDNA

[009] B domain-truncated human-porcine FVIII cDNA consisted of porcine Al and A3 domains, human signal peptide, A2, residual B (comprising the first 266 amino acids and eight glycosylation sites), CI and C2 domains (Sivalingam et al, 2014, also depicted in SEQ ID NO. 3. Overlap PCR was used in domain assembly (see Fig 18). A 1 and A3 domains were obtained by RT-PCR of total pig liver RNA based on the reference porcine cDNA sequence (NM_214167.1 ). Human domains were amplified from complete human FVIII cDNA in pSP64- VIII (American Type Culture Collection).

[0010] Restriction fragment length polymorphism

[0011] Restriction fragment length polymorphism (RFLP) was used to quantify site- specific integration of pZDonor- AA VS 1. Two hundred ng of genomic DNA was extracted from cells 4 days after electroporation of 10 μg pZDonor- AA VS 1 in the absence or presence of AAVS1 ZFN. PCR primers amplified a 1.9-kb region spanning the AAVSI integration site. Amplicons digested with HmdIII were resolved by electrophoresis in 8% polyacrylamide gels, post-stained with ethidium bromide and imaged (BioRad®Gel Doc 2000 transilluminator). Two bands (1- and 0.9-kb) indicated donor integration, while a single 1.9-kb band indicated no integration. The intensity and volume of DNA bands were quantified by Quantity One software (Bio-Rad).

[0012] CEL-1 assay

[0013] ZFN activity was assayed by the presence of non-homologous end-joining repair detected by CEL-1 nuclease using reagents and instructions of the Surveyor™ mutation detection kit (Transgenomic).

[0014] Flow cytometry

[0015] Transfection efficiencies were evaluated 24 hours after electroporation by fluorescence-activated cell analysis of GFP-expressing cells (BD FACSCalibur™ flow cytometer; 488 nm argon laser; 530/30 bandpass filter).

[0016] DNA damage response was assessed by histone Η2ΑΧ phosphorylation. CLECs were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS)/90% methanol 2 days after electroporation, permeabilized (0.5% Triton-X100, 2% bovine serum albumin) for 10 minutes and incubated with Alexa Fluor® 647-conjugated-anti-phosphohistone H2AX antibody (Serl39) (1:40 dilution; Cell Signaling Technology) for 1 hour at 25°C. Cells were washed twice with PBS, resuspended in 500 μΐ PBS and filtered through a 40 μπι nylon mesh. Flow cytometry was performed with a 633 nm He-Ne laser and 661/16 bandpass filter.

[0017] Data were analyzed using FlowJo v7.22 (FLOWJO).

[0018] Gene transfer

[0019] Two million K562 cells suspended in 100 μΐ Amaxa® Cell Line Nucleofactor Kit V solution containing 10 μg donor DNA plasmid and 5 μg ZFN plasmid or 5 μΐ AAVS1 ZFN mPvNA (Sigma- Aldrich) were electroporated using the TO 16 setting in Amaxa® Nucleofector I (Lonza). Where indicated, cells were co-transfected with pEGFP to assess efficiency of gene transfer. Stably integrated cells were selected by culture in G418 (0.8 mg/ml) for 14 days.

[0020] Two to 10 million primary human cells were electroporated (Amaxa® 4D Nucleofector; setting CM113) with 10 μg donor DNA plasmid and 5 μg ZFN plasmid, as indicated in the figure legends. Stably integrated cord-lining epithelial cells were resistant to puromycin (0.5 mg/ml for 7 days) (puro-CLECs). Wt-CLECs were untreated with plasmids.

[0021] FVIII assay

[0022] FVIII activity in overnight conditioned media of wt- and puro-CLECs was assayed using the Coatest SP FVIII kit (Chromogenix) and recommended protocol. Data are expressed as mUnits FVIII /million cells/24 hours (mean ± SD of triplicates).

[0023] Integration junction PCR

[0024] Integration junction PCR was performed on 200 ng genomic DNA using DyNAzyme EXT DNA polymerase (Thermo Scientific) and primers specific to the integrated vector and genomic sequences immediately adjacent to the integration site. Positive control PCR amplified a separate region within the AAVS1 locus 2 kb away from the integration site. Integration junction PCR products were sequenced to verify their identity. Overlapping long PCR and sequencing confirmed integration of the complete hybrid FVIII transgene.

[0025] Digital droplet PCR

[0026] Fifty ng genomic DNA from wt- and puro-CLECs, 1 μΜ of each primer and 0.25 μΜ BHQ1-FAM probes (Sigma- Aldrich) were added to QX200™ dd PCR™ supermix (Bio- Rad) in a final reaction volume of 20 μΐ and transferred to QX100™ Droplet Generator followed by 40 cycles of PCR amplification (annealing temperature 61°C and extension time 1 min. per cycle) using CI 000 Touch™ Thermal Cycler. Droplet PCR products were read on a QX100™ Droplet Reader and data analyzed using QuantaSoft™ software. Control 1 (forward: cctgccttaaacccagccag (SEQ ID NO: 17); reverse: atgacctcatgctcttggccctcgta (SEQ ID NO: 18; probe: aaccaccccagcagatactct, SEQ ID NO: 6) and control 2 (forward: tcccctcccagaaagacctgc; SEQ ID NO: 19, reverse: tcccctcccagaaagacctgc; SEQ ID NO: 20; probe: tacctaacgcactcctgggtga, SEQ ID NO: 21) amplified a genomic locus in human chromosome 19q 13.42. Reactions designed for the left (genome specific forward: ttcgggtcacctctcactcc; SEQ ID NO: 22, vector specific reverse: gacgcgcgtgaggaagagttc (SEQ ID NO: 23; probe: aggcgcaccgtgggcttgt, SEQ ID NO: 24) and right (vector specific forward: ctgtggaatgtgtgtcagttag, SEQ ID NO: 25; genome specific reverse: ggctccatcgtaagcaaacc, SEQ ID NO: 26; probe:cgcctctgcctctgagctat, SEQ ID NO: 27) integration junctions amplified AAVSl site- specific integrated vector while all integrated vectors regardless of genomic location were detected by vector specific PCR (forward: cgagatgaccgagtacaag; SEQ ID NO: 28; reverse: gctcgtagaaggggaggttg; SEQ ID NO: 29; probe: tcaccgagctgcaagaact, SEQ ID NO: 30). Each PCR reaction was performed in quadruplicate. Data are mean ± SD.

[0027] Amplicon deep sequencing

[0028] The predicted 10 most likely potential off-target sites previously published for AAVS 1 ZFNs (Hockemeyer et al, 2009) (OT1- OT10; Fig. 19) were investigated by massively parallel paired-end sequencing (MiSeq, Illumina) of amplicons from the AAVSl integration site and OT1 - OT10 of wt- and puro-CLECS. A commercially synthesized DNA fragment (GenScript) similar to the AAVSl locus sequence except for a 5 -bp deletion between the ZFN binding half-sites was spiked into the wild-type AAVS 1 locus amplicon at molar ratios of 1 : 10, 1: 100, 1:500 and 1: 1000 to determine the sensitivity of indel detection (Figure 13). Library construction (Nextera^® XT DNA Sample Preparation Kit, Illumina) and sequencing were performed by AITbiotech Pte. Ltd., Singapore.

[0029] Whole-genome sequencing

[0030] WGS was performed by BGI (Shenzhen, China). Libraries with 500-bp inserts were prepared from 5 μg randomly fragmented genomic DNA from wt- and puro-CLECs and sequenced on HiSeq 2000 (Illumina). Adapter sequences, duplicate reads (marked using Picard tools; picard.sourceforge.net), low quality reads (more than half the bases in a read having a quality <5) and reads with >10% unknown bases were removed from the raw data. Paired-end clean reads (90 bases) from both samples were aligned to the reference human genome (hgl9) using Burrows-Wheeler Aligner (BWA) (Li & Durbin, 2009) and stored in BAM format files. Both samples were sequenced to 23x depth.

[0031] RNA-seq

[0032] Total RNA from wt- and puro-CLECs was qualitatively assessed by Bioanalyzer (Agilent) and quantified by Qubit® fluorometer (Life Technologies). Two μg of high quality RNA (RIN>9) was used for library preparation following the standard protocol (Illumina TruSeq RNA Sample Prep v2 kit). Briefly, poly-A mRNA was purified on oligo-dT magnetic beads. After purification, mRNA was fragmented ( 150-250bp) and converted into first-strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis was performed with DNA polymerase I and RNaseH. cDNA fragments were next blunt-ended, a single "A" base added for ligation to indexed adapters with complementary T-overhangs. The indexed products were purified and enriched with PCR to create the final cDNA library. Indexed libraries were validated for size and purity by Bioanalyzer, and quantified using Quant-iT™ PicoGreen® dsDNA assay kit (Life Technologies). Libraries were normalized to 10 nM by real-time PCR (iTaq™ Universal SYBR® Green Supermix; BioRad) and equal volumes were pooled. Pooled libraries were denatured and diluted to 20 pM for clustering on the cBot before loading on the HiSeq 2000 to generate paired-end reads of 76bp.

[0033] Bioinformatic data analyses

[0034] Detection of on-target indels

[0035] All reads of the puro-CLEC sample were aligned against a 980-bp sequence containing the intended ZFN cleavage site with e-value cutoff 0.00001. All aligned reads were mapped back to the reference human genome (hgl9) using SMALT (www.sanger.ac.uk/resources/software/smalt/) to generate a sequence alignment file. VarScan v2.3.6 was used to detect indels using command "pileup2indel" (Koboldt et al, 2012).

[0036] Detection of off-target indels

[0037] All wt- and puro-CLEC reads aligned to hgl9 were analyzed for somatic variants using VarScan v2.3.6. The sequence alignments in binary alignment format were transformed to SAMtools pileup format using SAMtools (Li et al, 2009). The resulting pileup files were submitted for somatic variant calling by VarScan using the command "somatic" taking wt-CLEC as "normal" and puro-CLEC as "tumor" (Java -jar VarScan.jar somatic <pileup file of WT sample> <pileup file of PURO sample> output). Somatic variants identified by VarScan were further classified as high-confidence (.he) or low-confidence (.lc) using the command "processSomatic" (Java -jar VarScan.jar processSomatic output.indel). The high-confidence somatic variants were analyzed further to generate the final list of candidate indels for experimental validation.

[0038] Putative off-target sites in hg 19 were identified in silico using ZFN-Site (Cradick et al, 2011). The left and right ZFN half-sites were ATCCTGTCCCTA (SEQ ID NO: 31) and ACCCCACAGTGG (SEQ ID NO: 32), respectively. The allowed spacing was 5 or 6 bp and the maximum number of mismatches per half-site was set to 2. There was no overlap between high- confidence somatic indels and in silico predicted AAVS1-ZFN off-target sites.

[0039] Next, another method was adopted to identify possible off-target effects. Using an in-house Perl script, the reads were scanned for hot spot genomic regions in which multiple overlapping high-confidence somatic indel variants mapped. These genomic loci were subjected to experimental validation by PCR-Sanger sequencing.

[0040] Identification of AAVS1 ZFN-mediated insertion of FVIII donor DNA

[0041] All puro-CLEC reads were aligned to AAVS 1 sequences modified by integration of FVIII donor DNA and the reference human genome (hgl9). This identified prokaryotic sequences in the vector that were specific to FVIII donor DNA integration. The absence of these prokaryotic sequences in wt-CLEC sample reads and their presence only in puro-CLEC sample confirmed integration of FVIII donor DNA. To identify off-target integrations, reads were identified from puro-CLEC that were combinations of both human and non-human sequences (hereafter called mixed reads). Mixed reads were mapped to the reference human genome. This defined the locations of putative off-target integrations and simultaneously confirmed the absence of non-human sequences in hgl9. Only reads that aligned to the modified AAVS1 region were considered to be on-target ZFN-mediated insertions of FVIII donor DNA in the AAVS1 locus.

[0042] Detection of structural variants

[0043] As SVDetect (version r0.08; http://svdetect.sourceforge.net) (Zeitouni et al, 2010) and BreakDancer (version 1.4.4; http://breakdancer.sourceforge.net/) (Chen et al, 2009) require only abnormally mapped reads, the raw reads were first pre-processed with S AMtools (version 0.1.19; http://samtools.sourceforge.net ) and Picard. The command line used for SAMtools was: samtools view -b -h -F 10 -q 22 input.bam > output.bam.

[0044] This command retained in output.bam only reads which were abnormally mapped (mapped to different chromosomes and on different strands) and which had a minimum mapping quality of 22. The resulting bam file was further filtered to discard duplicate reads using the following command in Picard tools:

java -jar /opt/picard-l.l l l/MarkDuplicates.jar INPUT=output.bam OUTPUT=output.nodup.bam REMOVE_DUPLICATES=true AS SUME_S ORTED=true METRICS_FILE=metrics.output.txt.

[0045] The resulting bam files were submitted to SVDetect and BreakDancer for calling structural variants (SV). SVDetect and BreakDancer were both configured to detect rearrangements with 2 or more supporting read pairs using 8 times the standard deviation as threshold. After the first step of SVDetect, the resulting '.links' file containing all the called SVs was filtered for "imperfect duplicates" (as defined by Mijuskovic et al, 2012) with in-house Perl script. Links supported solely by clusters of imperfect duplicates were removed. However, links which had only some imperfect duplicates were preserved after removing the supporting imperfect duplicates. Following this, the files were further filtered by SVDetect' s own filtering process. The next step was to compare the filtered SVs called for both wt- and puro-CLECs. The option for comparing only the same SV type was turned off. No filtering of imperfect duplicates was done for BreakDancer output as this is not necessary if the anchoring region is set to 3 and the default value in BreakDancer is 7.

[0046] The results of BreakDancer and SVDetect were subjected to a final filter to identify overlaps with repetitive DNA and low mappability regions (Mijuskovic et al, 2012). SVs that were only supported by reads that overlapped into any of these regions were removed. Filtering was done with in-house Perl script and BEDtools (version 2.17.0; http://bedtools.readthedocs.org/en/latest/) (Quinlan & Hall, 2010). BED files of these regions needed for filtering were extracted as described (Mijuskovic etal, 2012) from the UCSC genome browser (http://genome.ucsc.edu/). In the final step, outputs from BreakDancer and SVDetect were inspected for genomic loci that had clusters of reads consistent with inter- or intra- chromosomal SVs specific to puro-CLECs. This was done with in-house Perl script and different degrees of freedom (DF) of clustering. The maximum DF was set to 3 i.e. genomic coordinates that differed by up to 999 bases were still considered as evidence of clustering.

[0047] RNA-Seq

[0048] Sequence reads were mapped to the reference human genome (hg 19) using TopHat (tophat.cbcb.umd.edu). Differential expression was calculated using Cufflinks (cufflinks.cbcb.umd.edu). Transcripts whose expression levels differed by >2-fold in puro- CLECs compared to wt-CLECs were considered significantly altered. DAVID (Database for Annotation, Visualization and Integrated Discovery) 2.1 Functional Annotation Tool (http://david.abcc.ncifcrf.gov) (Huang et al, 2009) was used to annotate significantly altered transcripts and for pathway mapping. Altered transcripts were also referenced to an aggregated compilation of oncogenes and tumor suppressor genes

(http://www.bushmanlab.org/links/genelists).

[0049] Validation of potential indels and structural variants

[0050] Genomic DNAs from wt- and puro-CLECs were amplified with phi29 polymerase (REPLI-g kit; Qiagen). High confidence indels were investigated by PCR-Sanger sequencing. Predicted unbalanced structural variants were investigated by quantitative PCR of genomic breakpoint regions identified by SVDetect and BreakDancer. Each 15μ1 reaction, performed in triplicate, comprised 30 ng genomic DNA and 0.3 μΜ of each primer in iTaq™ Universal SYBR^® Green Supermix (Bio-Rad). β- Actin amplification served as the internal control in each experiment. Fifty amplification cycles were run on CFX96 Touch™ Real-Time Detection System (Bio-Rad), after which melt curves confirmed product specificity and threshold cycle (CT) values were determined (CFX Manager™ software, Bio-Rad). The mean CT value of each test locus was normalized to its own actin CT value. Results are expressed as the ratio of normalized CT value of puro-CLEC genomic DNA normalized CT value of wt-CLEC genomic DNA at each putative breakpoint locus.

[0051] Sanger sequencing of indels

[0052] PCR amplicons of indels were sequenced using BigDye® chemistry in a 3730x1 sequencer (Life Technologies). Betaine was added to the sequencing reaction because of highly repetitive DNA sequences in indels 1 - 3. Indel 2 amplicon could not be obtained as a continuous sequence.

[0053] RT-PCR [0054] Quantitative RT-PCR was performed Fig. 3 to verify changes in the levels of PPP1R12C and selected transcripts in puro-CLECs. CLECs electroporated without plasmid DNA and of the same number of population doublings served as controls. Intron-spanning exonic primers were used to amplify the endogenous PPP1R12C transcript (exons 4-6), neighbouring genes within a 1-Mb interval centered on the AAVS1 integration site (LILRB4, ISOC2, PPP6R1, NAT14, ZNF579, FIZl and RDH13), potential interacting partners of PPP1R12C predicted by Gene Network Central™ (http://www.sabiosciences.com) and Human Protein-Protein Interaction Prediction (http://www.compbio.dundee.ac.uk) that were significantly altered by RNA-Seq analysis (DUSP1, DUSP6, CDC6 and DUSP16), and a housekeeping gene, GAPDH. Transcript levels were normalized to GAPDH expression and the fold-change in transcript levels in puro-CLECs was expressed relative to wt-CLECs using the 'delta-delta Q_T) method' (Livak & Schmittgen, 2001).

[0055] Cell proliferation assay

[0056] One hundred wt- or puro-CLECs in 100 μΐ culture medium were seeded into each of quadruplicate wells of a flat bottom 96-well tissue culture plate. MTS assay (CellTiter 96^® AQueous One Solution Cell Proliferation Assay; Promega) was performed after 7 days according to the recommended protocol. Absorbance at 490 nm was quantified using MRX II 96-well plate reader (Dynex).

[0057] Statistical analysis

[0058] Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc.). ANOVA and Tukey-Kramer tests were used to compare the means of three or more groups. Student's unpaired t test was used for comparison between two groups with equal variance and the Mann-Whitney test was used when variances were not assumed to be equal. P values < 0.05 were considered significant.

[0059] Concluding remarks

[0060] Problem

[0061] Serum-free recombinant FVIII protein administered prophylactically rather than only to halt bleeding episodes is the optimal treatment for patients with haemophilia A. As safe FVIII protein products are very costly, more than half the world's population of haemophilia A patients receive suboptimal therapy or even none at all. This has led to a lifetime of chronic disability for many thousands of patients. Autologous cell therapy is a potential solution. Using zinc finger nucleases (ZFNs), a FVIII transgene can be integrated into the genome of somatic cells ex vivo. These cells thereby acquire FVIII secretion and could function as a constant and durable source of FVIII after implantation in vivo. The safety of genome modification by ZFNs is a critical issue to address in clinical development. Unintended off-target genomic alterations have known oncogenic consequences. To date, off-target effects of ZFNs have been assessed using single techniques focused solely on genomic DNA. However, the observation that different methods do not identify the same off-target sites suggests that characterising ZFN-modified cells using a suite of different methods is preferable for assessing genotoxic and oncogenic risks.

[0062] Results

[0063] AAVS 1 ZFN were used to integrate a FVIII transgene in primary human umbilical cord-lining epithelial cells (CLECs). Several amino acid substitutions were combined to surprisingly enhance ZFN accuracy and activity (i.e. obligate heterodimerisation and enhanced DNA cleavage activity), and transient mild hypothermia was able to increase the efficiency of transgene integration. A gene trap strategy and selection for puromycin resistance was employed to favour on-target integration of the FVIII transgene, a B domain-truncated human-porcine hybrid which secreted higher FVIII activity than human FVIII. Under optimised conditions of AAVS 1 ZFN treatment, the complete FVIII transgene was integrated in CLECs which secreted robust amounts of FVIII. Quantitative genome copy number analysis of total FVIII donor DNA, left and right integration junctions of donor DNA in the AAVSl locus was consistent with monoallelic transgene integration and absence of significant off-target integrations. This was supported by quantitative RT-PCR which showed a reduction by half of PPP1R12C expression, the gene disrupted by FVIII transgene integration. Targeted deep sequencing of in silico predicted ten most likely off-target sites showed a low frequency of microdeletions only in an 8q24 intergenic locus. Whole-genome sequence data analysis suggested three potential indels. All three were intergenic; two were experimentally sequenced and shown to be a single base substitution or microsatellite DNA expansions. Four unbalanced chromosomal rearrangements suggested by whole-genome sequence analysis were not validated by experimental genome copy number analysis and absence of the predicted abnormal chromosomal junctions. RNA-seq was used to provide a functional readout of off-target genomic events. This revealed a very small footprint in the transcriptome of ZFN-mediated FVIII integration in the AAVSl locus. Of 17,751 total transcripts, only 90 (0.5%) had levels that were altered by 2-fold or more. Pathway analysis did not map the dysregulated transcripts to any canonical oncogenic pathway but only to chemokine-chemokine receptor interactions, consistent with a known function of PPP1R12C. Broader analysis of RNA-seq data to probe possible consequences of PPP1R12C haploinsufficiency was uniformly negative except for increased expression of only DUSP6, whose known effects are antiproliferative and antioncogenic. This was supported by the slightly lower proliferation rate of ZFN-modified CLECs. AAVS 1 ZFN was also active in primary adult dermal fibroblasts, bone marrow- and adipose-derived stromal cells. Under the same conditions which integrated FVIII transgene in CLECs, these adult cells could also be modified to integrate and secrete FVIII.

[0064] Impact

[0065] The present invention shows for the first time that AAVS 1 ZFN and FVIII donor DNA with design features that favour accurate and efficient on-target integration can modify human neonatal and adult primary somatic cells to secrete FVIII without incurring significant off-target alterations that could portend oncogenic risk. These results suggest that AAVS 1 ZFN- mediated FVIII transgene integration for autologous cell therapy of haemophilia A is unlikely to cause adverse complications and is thus a very promising approach that can be further developed for clinical therapy.

[0066] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.

[0067] Comparison of hybrid FVIII amino acid sequence

[0068] (Top sequence) Hybrid FVIII_KON lab = 1709 aa (retained 266 aa of B-domain with 8 glycosylation sites, SEQ ID NO: 4

[0069] (Bottom sequence) Hybrid FVI I I_Doering lab = 1467 aa (SEQ ID NO: 5, described in Doerin et al, Molecular Therapy vol. 17 no. 7, 1145-1154 July 2009

[0070] Query: . /wwwtmp/lalign29380.1. seq

l>>>Hybrid FVIII_KON lab 1709 bp - 1709 aa

Library : . /wwwtmp/lalign29380.2. seq

1467 residues in 1 sequences

[0071] Statistics: (shuffled [500]) MLE statistics: Lambda= 0.1781; K=0.02504

statistics sampled from 1 (1) to 500 sequences

[0072] Threshold: E() < 10 score: 49

[0073] Algorithm: Smith-Waterman (SSE2, Michael Farrar 2006) (7.2 Nov 2010)

[0074] Parameters: BL50 matrix (15:-5), open/ext: -12/-2

[0075] Scan time: 0.180

[0076] >>Hybrid FVIII_Doering lab 1467 bp

(1467 aa)

[0077] Waterman-Eggert score: 9322; 2400.4 bits; E(l) < 0 84.1% identity (85.2% similar) in 1709 aa overlap (1-1709:1- 1467)

10 20 30 40 50 60

Hybrid MQIELSTCFFLCLLRFCFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL

Hybrid MQLELSTCVFLCLLPLGFSAIRRYYLGAVELSWDYRQSELLRELHVDTRFPATAPGALPL

10 20 30 40 50 60 70 80 90 100 110 120

Hybrid GP SVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPT IQAEVYDTVWTLKNMASHPVSLHA

Hybrid GP SVLYKKTVFVEFTDQLFSVARPRPPWMGLLGPT IQAEVYDTVWTLKNMASHPVSLHA

70 80 90 100 110 120

130 140 150 160 170 180

Hybrid VGVSFWKS SEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS

Hybrid VGVSFWKS SEGAEYEDHTSQREKEDDKVLPGKSQTYVWQVLKENGPTASDPPCLTYSYLS

130 140 150 160 170 180

190 200 210 220 230 240

Hybrid HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM

Hybrid HVDLVKDLNSGLIGALLVCREGSLTRERTQNLHEFVLLFAVFDEGKSWHSARNDSWTRAM

190 200 210 220 230 240

250 260 270 280 290 300

Hybrid DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHS IFLEGHTFLVRH

Hybrid DPAPARAQPAMHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTSPEVHS IFLEGHTFLVRH

250 260 270 280 290 300

310 320 330 340 350 360

Hybrid HRQASLEI SPLTFLTAQTFLMDLGQFLLFCHI SSHHHGGMEAHVRVESCAEEPQLRRKAD

Hybrid HRQASLEI SPLTFLTAQTFLMDLGQFLLFCHI SSHHHGGMEAHVRVESCAEEPQLRRKAD

310 320 330 340 350 360

370 380 390 400 410 420

Hybrid EEEDYDDNLYDSDMDWRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA

Hybrid EEEDYDDNLYDSDMDWRLDGDDVSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLA

370 380 390 400 410 420

430 440 450 460 470 480

Hybrid PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL

Hybrid PDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTL

430 440 450 460 470 480

490 500 510 520 530 540

Hybrid LI IFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFP ILPGEIFKYKWTVTVEDGP

Hybrid LI IFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFP ILPGEIFKYKWTVTVEDGP

490 500 510 520 530 540

550 560 570 580 590 600

Hybrid TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE

Hybrid TKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE

550 560 570 580 590 600 610 620 630 640 650 660

Hybrid NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILS

Hybrid NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILS

610 620 630 640 650 660

670 680 690 700 710 720

Hybrid IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG

Hybrid IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG

670 680 690 700 710 720

730 740 750 760 770 780

Hybrid MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTI

Hybrid MTALLKVS SCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFAQNSRPPSA

730 740 750 760 770

790 800 810 820 830 840

Hybrid PENDIEKTDPWFAHRTPMPKIQNVSS SDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDP S

Hybrid

850 860 870 880 890 900

Hybrid PGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST

Hybrid

910 920 930 940 950 960

Hybrid SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE

Hybrid SAP

970 980 990 1000 1010 1020

Hybrid NNDSKLLESGLMNSQES SWGKNVSSTESGRLFKGKRAHGPALLTKDGRTERLCSQNPPVL

Hybrid -KPPVL

1030 1040 1050 1060 1070 1080

Hybrid KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFD IYDEDENQSPRSFQKRTRHYF IAAV

Hybrid RRHQRDISLPTFQPEEDKMDYDD IFSTETKGEDFD IYGEDENQDPRSFQKRTRHYF IAAV

780 790 800 810 820 830

1090 1100 1110 1120 1130 1140

Hybrid EQLWDYGMSESPRALRNRAQNGEVPRFKKWFREFADGSFTQPSYRGELNKHLGLLGPYI

Hybrid EQLWDYGMSESPRALRNRAQNGEVPRFKKWFREFADGSFTQPSYRGELNKHLGLLGPYI 840 850 860 870 880 890

1150 1160 1170 1180 1190 1200

Hybrid RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM

Hybrid RAEVEDNIMVTFKNQASRPYSFYSSLISYPDDQEQGAEPRHNFVQPNETRTYFWKVQHHM 900 910 920 930 940 950

1210 1220 1230 1240 1250 1260

Hybrid APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF

Hybrid APTEDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRANTLNAAHGRQVTVQEFALFFTIF 960 970 980 990 1000 1010

1270 1280 1290 1300 1310 1320

Hybrid DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY

Hybrid DETKSWYFTENVERNCRAPCHLQMEDPTLKENYRFHAINGYVMDTLPGLVMAQNQRIRWY 1020 1030 1040 1050 1060 1070

1330 1340 1350 1360 1370 1380

Hybrid LLSMGSNENIHS IHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLP SKVGIWRIECLIG

Hybrid LLSMGSNENIHS IHFSGHVFSVRKKEEYKMAVYNLYPGVFETVEMLP SKVGIWRIECLIG 1080 1090 1100 1110 1120 1130

1390 1400 1410 1420 1430 1440

Hybrid EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS INAWS

Hybrid EHLQAGMSTTFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS INAWS 1140 1150 1160 1170 1180 1190

1450 1460 1470 1480 1490 1500

Hybrid TKEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQF I IMYSLDGKKWQTYRGNSTGTL

Hybrid TKEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYI SQF I IMYSLDGKKWQTYRGNSTGTL 1200 1210 1220 1230 1240 1250

1510 1520 1530 1540 1550 1560

Hybrid MVFFGNVDSSGIKHNIFNPP I IARYIRLHPTHYS IRSTLRMELMGCDLNSCSMPLGMESK

Hybrid MVFFGNVDSSGIKHNIFNPP I IARYIRLHPTHYS IRSTLRMELMGCDLNSCSMPLGMESK 1260 1270 1280 1290 1300 1310

1570 1580 1590 1600 1610 1620

Hybrid AI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV

Hybrid AI SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGV 1320 1330 1340 1350 1360 1370

1630 1640 1650 1660 1670 1680

Hybrid TTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT Hybrid TTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT 1380 1390 1400 1410 1420 1430

1690 1700

Hybrid RYLRIHPQSWVHQIALRMEVLGCEAQDLY

Hybrid RYLRIHPQSWVHQIALRMEVLGCEAQDLY

1440 1450 1460

[0078] Scientific References cited

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Claims

What is claimed is:

1. A method of generating a mammalian stem cell carrying a transgene, the method comprising inserting a transgene into the genome of the mammalian stem cell by means of zinc finger nuclease (ZFN) mediated integration.

2. The method of claim 1 , wherein the stem cell is selected from the group consisting of a stem cell isolated from the amniotic membrane of the umbilical cord (cord lining stem cell), a stem cell isolated from Wharton's Jelly of the umbilical cord, a stem cell isolated from the amniotic membrane of the placenta, a stem cell isolated from the endothelium or the subendothelial layer of the umbilical cord vein, and a stem cell isolated from umbilical cord blood.

3. The method of claim 2, wherein the mammalian stem cell is selected from the group consisting of a human stem cell, a canine stem cell, a feline stem cell, an equine stem cell, a stem cell of an ape, or a stem cell of a macaque.

4. The method of claim 2 or 3, wherein the cord lining stem cell is an epithelial or a mesenchymal stem cell.

5. The method of any of claims 2 to 4, wherein the transgene is integrated into the AAVS 1 locus (present on human chromosome 19 ql3.3-qter) of a human cord lining stem cell.

6. The method of any of the foregoing claims, wherein the method comprises inserting the transgene by means of a mutated zinc finger nuclease.

8. The method of claim 7, wherein the zinc finger nuclease comprises at least one mutation in the Fokl cleavage domain Fokl, to provide a variant with enhanced cleavage activity.

9. The method of claim 8, wherein the mutated zinc finger nuclease comprises the S418P and K441E substitutions in both the right and left monomer at positions corresponding to positions 418 and 441 of wildtype Fokl of sequence of SEQ ID NO. 6.

10. The method of any of claims 1 to 9, wherein the zinc finger nuclease is a heterodimer.

11. The method of claim 8 or 9, wherein the catalytic domain of the Sharkey variant has the amino acid sequence of any one of SEQ ID NOs. 9, 12 or 13.

12. The method of any of claims 8 or 10, wherein the mutated zinc finger nuclease comprises the amino acid substitutions S418P, K441E and H537R in the right Fokl monomer and the amino acid substitutions S418P, K441E and N496D in the left Fokl monomer, and wherein the positions correspond to the respective positions of SEQ ID NO. 6.

13. The method of any of claims 8 to 12, wherein the mutated zinc finger nuclease (obligate heterodimer) comprises the two amino acid substitutions E490K and I538K in the Fokl monomer fused to the right AAVS 1 homology arm and the two amino acid substitutions Q468E and 1499 L in the monomer fused to the left homology arm, and wherein the positions correspond to the respective positions of SEQ ID NO. 6.

14. The method of any of the foregoing claims, where the zinc finger nuclease comprises a zinc finger protein selected from the group of a Cys2His2-like zinc finger protein have the amino acid sequence motif X2-Cys-X2,4-Cys-Xi2-His-X3,4,5-His, a Gag-knuckle zinc finger protein Treble- clef, a zinc ribbon zinc finger protein and a Zn2/Cys6 zinc finger protein.

15. The method of any of the foregoing claims wherein the zinc finger protein is selected from the group consisting of a P3 zinc finger protein, an E2C (E6) zinc finger protein, an E5 zinc finger protein, an E4 zinc finger protein and an E3 zinc finger protein.

16. The method of any of claims 7 to 15, wherein the integration reaction is carried out at a temperature range between about 25°C and about 32°C.

17. The method of claim 16, wherein the integration reaction is carried out at a temperature of about 30°C.

18. The method of any of claims 7 to 17, wherein the integration of the transgene is carried out by transfection.

19. The method of claim 18, wherein the transfection is carried out using a single plasmid that delivers both monomers of the zinc finger nuclease, preferably of the Sharkey or Enhanced Sharkey AAVS1 zinc finger nuclease monomers.

20. The method of claim 18 or 19, wherein the transfection is carried out using transient hypothermia.

21. The method of any of the foregoing claims, wherein the transgene is selected from the group of nucleic acid molecule (gene) encoding a blood coagulation factor and a nucleic acid molecule (gene) encoding a protein hormone secreted by an endocrine gland.

22. The method of claim 21, wherein the blood coagulation factor is selected from the group consisting of factor VII, factor VIII and factor IX.

23. The method of claim 21, wherein a deficiency of the expression or secretion of the protein hormone secreted by an endocrine gland is associated with an endocrine deficiency.

24. The method of claim 23, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

24. The method of claim 21, wherein the transgene is a nucleic acid molecule encoding a chimeric factor VIII polypeptide.

25. The method of claim 24, wherein the transgene encodes a chimeric factor VIII polypeptide comprising human and porcine domains.

26. The method of claim 25, wherein the transgene encodes a chimeric factor VIII polypeptide comprising or consisting of porcine Al and A3 domains, human signal peptide, the human A2 domain, a residual human B domain and human CI and C2 domains and/or wherein the transgene comprises a sequence of SEQ ID NO. 3.

27. The method of claim 26, wherein the residual human B domain comprises the first 266 amino acids and eight glycosylation sites.

28. The method of claim 26 or 27, wherein the transgene encodes the chimeric factor VIII polypeptide of SEQ ID NO: 4 or a polypeptide having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92%, 93%, 94%, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of polypeptide of SEQ ID NO. 4.

29. A mammalian stem cell carrying a transgene obtained by a method as defined in any of claim 1 to 28.

30. The mammalian stem cell of claim 29, wherein the stem cell is selected from the group consisting of a stem cell isolated from the amniotic membrane of the umbilical cord (cord lining stem cell), a stem cell isolated from Wharton's Jelly of the umbilical cord, a stem cell isolated from the amniotic membrane of the placenta, a stem cell isolated from the endothelium or the subendothelial layer of the umbilical cord vein, and a stem cell isolated from umbilical cord blood.

31. The mammalian stem cell of claim 30, wherein the mammalian stem cell is selected from the group consisting of a human stem cell, a canine stem cell, a feline stem cell, an equine stem cell, a stem cell of an ape, or a stem cell of a macaque.

32. The mammalian stem cell of claim 30 or 31 , wherein the cord lining stem cell is an epithelial or a mesenchymal stem cell.

33. The mammalian stem cell of any of claims 30 to 32, wherein the transgene is integrated into the AAVSl locus (present on human chromosome 19 ql3.3-qter) of a human cord lining stem cell.

34. The mammalian stem cell of claim 33, wherein the transgene is a gene encoding a chimeric factor VIII polypeptide.

35. The mammalian stem cell of claim 34, wherein the transgene encodes a chimeric factor VIII polypeptide comprising human and porcine domains.

36. The mammalian stem cell of claim 35, wherein the transgene encodes a chimeric factor VIII polypeptide comprising or consisting of porcine Al and A3 domains, human signal peptide, the human A2 domain, a residual human B domain and human C 1 and C2 domains and/or wherein the transgene comprises a sequence of SEQ ID NO. 3.

37. The mammalian stem cell of claim 36, wherein the residual human B domain comprises the first 266 amino acids and eight glycosylation sites.

38. The mammalian stem cell of claim 36 or 37, wherein the transgene encodes the chimeric factor VIII polypeptide of SEQ ID NO. 4 or a polypeptide having at least 85 %, 86, %, 87 %, 88%, 89 %, 90 %, 91 %, 92%, 93%, 94%, 95 %, 96 %, 97 %, 98 or 99 % sequence identity with the sequence of polypeptide of SEQ ID NO. 4.

39. The use of a mammalian stem cell as defined in any of claims 29 to 38 for treating a disease.

40. The use of claim 39, wherein the disease is a disease associated with a deficiency of a gene or deficiency of the expression of the gene, wherein the gene is selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland.

41. The use of claim 40, wherein the blood coagulation factor is selected from the group consisting of factor VII, factor VIII and factor IX.

42. The use of claim 40, wherein the disease is hemophilia.

43. The use of claim 42, wherein hemophilia is selected from the group consisting of hemophilia A, hemophilia B and hemophilia C.

44. The use of claim 39, wherein the disease is associated with an endocrine deficiency.

45. The use of claim 44, wherein the disease is associated with a deficiency of the expression or secretion of the protein hormone secreted by an endocrine gland.

46. The use of claim 45, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

47. A method of treating a patient having a disease, the method comprising administering the patient a mammalian stem cell as defined in any of claims 29 to 39.

48. The method of claim 47 of treating a patient, wherein the disease is a disease associated with a deficiency of a gene or deficiency of the expression of the gene, wherein the gene is selected from the group consisting of a gene encoding a blood coagulation factor and a gene encoding a protein hormone secreted by an endocrine gland.

49. The method of claim 48, wherein the blood coagulation factor is selected from the group consisting of factor VII, factor VIII and factor IX.

50. The method of claim 49, wherein the disease is hemophilia.

51. The method of claim 50, wherein hemophilia is selected from the group consisting of hemophilia A, hemophilia B and hemophilia C.

52. The method of claim 48, wherein the disease is associated with an endocrine deficiency.

53. The method of claim 52, wherein the disease is associated with a deficiency of the expression or secretion of the protein hormone secreted by an endocrine gland.

54. The method of claim 53, wherein the deficiency of the protein hormone is associated with an endocrine deficiency selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

55. The method of any of claims 47 to 54, wherein the mammalian stem cells are administered by implantation or injection.

56. The method of claim 55, wherein the stem cells are implanted subcutaneously.

57. A pharmaceutical composition containing a mammalian stem cells as defined in any of claims 29 to 38.

58. The pharmaceutical composition of claim 57 being adapted for implantation or injection.

59. The pharmaceutical composition of claim 58, being adapted for subcutaneous implantation.