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Supplementary figure S1 : Gene disruption analysis in HEK293 cells. Supplementary figure S2: Optimization of the generation of double KO cells Supplementary table S1: Deep sequencing analysis of TCR-negative cells Supplementary figure S3: Representative deletions found at disrupted TRAC gene Supplementary figure S4: Size distribution of the mutations found in TALEN-treated T cells Supplementary table S2: Size of insertions/deletions in mutated CD52/TRAC alleles Supplementary Table S3: NGS sequencing analysis of off target cleavage Supplementary figure S5: Gating strategy for definition of T-cellsubsets in UCART19

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator–like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19+ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an “off-the-shelf” manner akin to other biologic immunopharmaceuticals. Cancer Res; 75(18); 3853–64. ©2015 AACR.

Supplementary Methods from Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies

Summary Background Genome-edited donor-derived allogeneic anti-CD19 chimeric antigen receptor (CAR) T cells offer a novel form of CAR-T-cell product that is available for immediate clinical use, thereby broadening access and applicability. UCART19 is one such product investigated in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Two multicentre phase 1 studies aimed to investigate the feasibility, safety, and antileukaemic activity of UCART19 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Methods We enrolled paediatric or adult patients in two ongoing, multicentre, phase 1 clinical trials to evaluate the safety and antileukaemic activity of UCART19. All patients underwent lymphodepletion with fludarabine and cyclophosphamide with or without alemtuzumab, then children received UCART19 at 1·1–2·3 × 106 cells per kg and adults received UCART19 doses of 6 × 106 cells, 6–8 × 107 cells, or 1·8–2·4 × 108 cells in a dose-escalation study. The primary outcome measure was adverse events in the period between first infusion and data cutoff. These studies were registered at ClinicalTrials.gov, NCT02808442 and NCT02746952. Findings Between June 3, 2016, and Oct 23, 2018, seven children and 14 adults were enrolled in the two studies and received UCART19. Cytokine release syndrome was the most common adverse event and was observed in 19 patients (91%); three (14%) had grade 3–4 cytokine release syndrome. Other adverse events were grade 1 or 2 neurotoxicity in eight patients (38%), grade 1 acute skin graft-versus-host disease in two patients (10%), and grade 4 prolonged cytopenia in six patients (32%). Two treatment-related deaths occurred; one caused by neutropenic sepsis in a patient with concurrent cytokine release syndrome and one from pulmonary haemorrhage in a patient with persistent cytopenia. 14 (67%) of 21 patients had a complete response or complete response with incomplete haematological recovery 28 days after infusion. Patients not receiving alemtuzumab (n=4) showed no UCART19 expansion or antileukaemic activity. The median duration of response was 4·1 months with ten (71%) of 14 responders proceeding to a subsequent allogeneic stem-cell transplant. Progression-free survival at 6 months was 27%, and overall survival was 55%. Interpretation These two studies show, for the first time, the feasibility of using allogeneic, genome-edited CAR T cells to treat patients with aggressive leukaemia. UCART19 exhibited in-vivo expansion and antileukaemic activity with a manageable safety profile in heavily pretreated paediatric and adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. The results this study are an encouraging step forward for the field of allogeneic CAR T cells, and UCART19 offers the opportunity to treat patients with rapidly progressive disease and where autologous CAR-T-cell therapy is unavailable. Funding Servier.

Herpes simplex virus type 1 (HSV1) is a major health problem. As for most viral diseases, current antiviral treatments are based on the inhibition of viral replication once it has already started. As a consequence, they impair neither the viral cycle at its early stages nor the latent form of the virus, and thus cannot be considered as real preventive treatments. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as meganucleases. With the aim of a proof of concept study, we generated several meganucleases recognizing HSV1 sequences, and assessed their antiviral activity in cultured cells. We demonstrate that expression of these proteins in African green monkey kidney fibroblast (COS-7) and BSR cells inhibits infection by HSV1, at low and moderate multiplicities of infection (MOIs), inducing a significant reduction of the viral load. Furthermore, the remaining viral genomes display a high rate of mutation (up to 16%) at the meganuclease cleavage site, consistent with a mechanism of action based on the cleavage of the viral genome. This specific mechanism of action qualifies meganucleases as an alternative class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms.

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138-Lys139 and Lys142-Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.

International audience; Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator–like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19+ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an “off-the-shelf” manner akin to other biologic immunopharmaceuticals. Cancer Res; 75(18); 3853–64. ©2015 AACR.

Protoporphyrinogen oxidase, the last enzyme of the common branch of the heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides. These compounds inhibit the enzyme competitively with respect to the tetrapyrrole substrate, protoporphyrinogen IX. We used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenyleneiodonium cation, a known inhibitor of several flavoproteins. Diphenyleneiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidase competitively with molecular oxygen. The typical slow-binding kinetics suggested that the enzyme with a reduced flavin rapidly combined with the inhibitor to form an initial complex which then slowly isomerized to a modified enzyme-inhibitor complex (Ki = 6.75 x 10(-8) M, Ki* = 4.1 x 10(-9) M). This inhibition was strongly pH-dependent and was maximal at pH 8. Substituted diphenyleneiodoniums were synthesized and shown to be even better inhibitors than 2,2'-diphenyleneiodonium: Ki = 4.4 x 10(-8) M and Ki* = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyleneiodonium, Ki = 2.2 x 10(-8) M and Ki * = 1.1 x 10(-9) M for 6-methyl-2,2'-diphenyleneiodonium, and Ki = 6.4 x 10(-9) M and Ki* = 1.2 x 10(-1)2 M for 4-nitro-2,2'-diphenyleneiodonium. The 4-nitro-2,2'-diphenyleneiodonium was a quasi irreversible inhibitor (k5/k6 > 5000). Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inhibitors that act via a mechanism very different from that of diphenyl ether-type herbicides and appear to be promising tools for studies on the structure-function relationships of this agronomically important enzyme.

This review summarizes the overall downstream applications of currently available genome customization systems, based on cell-based assays (in cellulo) or animal models (in vivo). These include (i) functional genomics, (ii) drug discovery, (iii) bioproduction, (iv) cell transformation (i.e. immortalization, reprogrammation and differentiation), as well as (v) molecular biology and microbiology tools. The different enzymatic systems that exist to specifically modify (insertional or site-directed mutagenesis), modulate (knock-down) and disrupt (constitutive or conditional knock-out) cellular genes, or to integrate transgenic elements (knock-in) at specific chromosomal loci will be discussed herein.

Purpose Despite advances in antiviral therapies, past history of HSV keratitis is associated with poor prognosis of subsequent penetrating keratoplasty.The aim of this study is to assess the antiviral property of a meganuclease targeting HSV in the prevention of HSV endothelitis. Methods Normal rabbit corneas were placed in organ culture using an immersion method and transduced by a recombinant adeno-associated virus (rAAV) allowing constitutive expression of meganucleases targeting HSV-1 genome or containing the same expression cassette with a non-coding sequence. These organs were then submitted to infection by recombinant HSV-1 F(1) virus equipped with a LacZ expressing cassette at M.O.I. 0.001 to 0.1% in liquid or semi-solid medium. Infection rates for plaques or cells in endothelium were established by immunostaining of envelope protein gD or X-gal staining after the end of first or second lytic cycle. Results Meganuclease targeting the ICP0 gene which encodes an E3 ubiquitin ligase involved in viral reactivation and replication did not change infection rates in the present organ culture model, but reduced the average size of plaques in endothelium with a decrease of 27-46%. Conversely, the meganuclease directed against the major capsid protein UL19 lowered the number and size of plaques, both being reduced by half at M.O.I. 0.001%. Consequently, the expression of a meganuclease in endothelium, evidenced by RT-PCR, could either reduce infectious particle production or induce cell resistance to HSV-1. Conclusion These meganucleases are currently checked for their anti-infective properties in an in vivo model of endothelitis.

Purpose Current anti-viral treatments of Herpes simplex virus type 1 (HSV1) inhibit the viral replication but impair neither the viral cycle at its early stages nor the latent form of the virus. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as specific meganucleases. Methods Co-transfections with plasmid expressing I-CreI variants cleaving target sequences and GFP were performed using Lipofectamine on BSR cells for 48 hours. After infection for 8h with SC-16 at different MOIs (Multiplicity Of Infection) ranging from 0.1 to 8, cells were incubated with i) HSV1 rabbit antibodies and ii) with phycoerytrine (PE) conjugated goat anti-rabbit IGg secondary antibodies. Fluorescence-activated cell sorting (FACS) analysis was performed. The ratio of infected cells among transfected (GFP+) and non transfected (GFP-) cells were counted and an antiviral index was calculated. Results GFP+ and GFP- cells were both well represented in all experiments, with an average GFP+ cell frequency of 0.63 over 90 transfections (standard deviation: 0.20). A strong inhibitory effect was observed at MOIs of 0.1 and 0.5, with 4 to 7 times less infection among GFP+ cells than among GFP- ones. This effect was however not detected by a MOI of 2. Conclusion Meganuclease proteins in BSR cells inhibits the replication of HSV1, at moderate MOIs, inducing a significant reduction of the viral particules. This qualifies meganucleases as a new class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms. Specific meganucleases may thus be used in the future with the aim of controlling recurrent herpetic keratitis. Commercial interest

Meganucleases (MNs) are highly specific enzymes that can induce homologous recombination in different types of cells, including mammalian cells. Consequently, these enzymes are used as scaffolds for the development of custom gene-targeting tools for gene therapy or cell-line development. Over the past 15 years, the high resolution X-ray structures of several MNs from the LAGLIDADG family have improved our understanding of their protein-DNA interaction and mechanism of DNA cleavage. By developing and utilizing high-throughput screening methods to test a large number of variant-target combinations, we have been able to re-engineer scores of I-CreI derivatives into custom enzymes that target a specific DNA sequence of interest. Such customized MNs, along with wild-type ones, have allowed for exploring a large range of biotechnological applications, including protein-expression cell-line development, genetically modified plants and animals and therapeutic applications such as targeted gene therapy as well as a novel class of antivirals.

Homing endonucleases or 'meganucleases' are a type of very rare-cutting endonuclease. They recognize much larger DNA sequences than do 'classical' restriction enzymes, so they produce a low frequency of cleavage in the human genome. This makes them of interest for gene therapy, as highly specific molecular scalpels to target specific genes. In a proof-of-principle experiment, Redondo et al. have engineered homing endonucleases with the potential to aid the repair of the gene mutated in xeroderma pigmentosum, a disorder that affects the nucleotide excision repair machinery, compromising the body's ability to remove damage caused by ultraviolet light. Xeroderma pigmentosum patients have high frequencies of mutations and hence a high predisposition to develop skin cancer. The newly designed enzymes, called Amel3–4 and Ini3–4, are derivatives of the homing endonuclease I-CreI that cleave the human XPC gene both in vitro and in vivo. Structural analysis suggests that their catalytic mechanism of cleavage is similar to that of the wild type homodimeric I-CreI, and both Amel3–4 and Ini3–4 induce high levels of gene targeting in mammalian cells. This work demonstrates a gene repair technology with the potential to repair genes in xeroderma pigmentosum that produce other monogenetic diseases. Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers1. The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

Meganucleases are sequence-specific endonucleases which recognize large (>12 bp) target sites in living cells and can stimulate homologous gene targeting by a 1000-fold factor at the cleaved locus. We have recently described a combinatorial approach to redesign the I-CreI meganuclease DNA-binding interface, in order to target chosen sequences. However, engineering was limited to the protein regions shown to directly interact with DNA in a base-specific manner. Here, we take advantage of I-CreI natural degeneracy, and of additional refinement steps to extend the number of sequences that can be efficiently cleaved. We searched the sequence of the human XPC gene, involved in the disease Xeroderma Pigmentosum (XP), for potential targets, and chose three sequences that differed from the I-CreI cleavage site over their entire length, including the central four base-pairs, whose role in the DNA/protein recognition and cleavage steps remains very elusive. Two out of these targets could be cleaved by engineered I-CreI derivatives, and we could improve the activity of weak novel meganucleases, to eventually match the activity of the parental I-CreI scaffold. The novel proteins maintain a narrow cleavage pattern for cognate targets, showing that the extensive redesign of the I-CreI protein was not made at the expense of its specificity. Finally, we used a chromosomal reporter system in CHO-K1 cells to compare the gene targeting frequencies induced by natural and engineered meganucleases. Tailored I-CreI derivatives cleaving sequences from the XPC gene were found to induce high levels of gene targeting, similar to the I-CreI scaffold or the I-SceI “gold standard”. This is the first time an engineered homing endonuclease has been used to modify a chromosomal locus.

3069 Background: Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are of high potential in treating different kinds of cancer. However,...

Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cell's potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab.We have obtained proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and cD19-CAR+ T-cells (UCART19) using a good manufacturing practice-compatible process and have demonstrated that the resulting UCART19 cells were functional using in vitro assays. Furthermore we have demonstrated the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Finally, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled.

Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities.

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN™), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cell’s potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to coexpress the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. We previously provided proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and CD19-CAR+ T-cells (UCART19) using a good manufacturing practice–compatible process, and we also demonstrated that the resulting UCART19 cells were functional using in vitro assays. Here we report the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. In conclusion, our work significantly enlarges upon previous results by showing in vivo that (1) concomitant inactivation of a second gene has no deleterious effects on T-cells, (2) the antitumor potency of manufactured TCR/CD52-deficient CD19–CAR+ T-cells is similar to that of standard CD19-CAR+ T-cells, (3) TCR gene inactivation is efficient at preventing potential graft-versus-host reaction, and (4) allogeneic T-cells can be depleted by the use of rituximab. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled. Disclosures Gouble: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Derniame:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Desseaux:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have established a highly efficient, 18-day, good manufacturing practice (GMP)–compatible process to produce CAR T-cells from healthy donor peripheral blood mononuclear cells (PBMCs). To circumvent the potential of allogeneic T-cells inducing graft-versus-host disease (GvHD) in recipient patients, the TCR alpha constant (TRAC) gene was inactivated using a proprietary transcription activator-like effector nuclease (TALEN™)-mediated gene editing technology. The CD52 gene was also disrupted using another specific TALEN™ to allow the administration of engineered T-cells following an alemtuzumab-based lymphodepleting therapy. The antitumor activity of these double-knockout CAR T-cells was shown to be as potent as non-nuclease-edited cells expressing the same CAR in vitro. The current manufacturing process is highly reproducible, making it suitable for use in a larger scale manufacturing platform for administration as “off-the-shelf” immunopharmaceuticals. We estimate that a single production run, starting from a healthy volunteer leukapheresis product containing 109 PBMCs, would allow the production of up to 500 doses of CAR double-knockout T-cells, at 2x107 cells per dose, allowing the extension of CAR therapies to a larger number of patients. Our results provide the proof of concept for the general applicability of this approach as a platform for large-scale GMP–compliant manufacturing of allogeneic, off-the-shelf, non-alloreactive, frozen CAR T-cells. From this manufacturing platform, we produced UCART19 cells, which are TCR/CD52-deficient, RQR8+ (as a safety attribute), and anti-CD19 CAR+, to investigate their potential in the treatment of CD19+ B cell leukemias. We believe this adaptable manufacturing platform offers multiple opportunities to improve CAR T-cell therapies through multiplex genome editing, such as rendering UCART cells resistant to standard chemotherapy or to tumor evasion mechanisms. Disclosures Derniame: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Beurdeley:Cellectis SA: Employment. Reynier:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.

Protein acylation is an important way in which a number of proteins with a variety of functions are modified. The physiological role of the acylation of cellular proteins is still poorly understood. Covalent binding of fatty acids to nonintegral membrane proteins is thought to produce transient or permanent enhancement of the association of the polypeptide chains with biological membranes. In this paper, we investigate the functional role for the palmitoylation of an atypical membrane-bound protein, yeast protoporphyrinogen oxidase, which is the molecular target of diphenyl ether-type herbicides. Palmitoylation stabilizes an active heat- and protease-resistant conformation of the protein. Palmitoylation of protoporphyrinogen oxidase has been demonstrated to occur in vivo both in yeast cells and in a heterologous bacterial expression system, where it may be inhibited by cerulenin leading to the accumulation of degradation products of the protein. The thiol ester linking palmitoleic acid to the polypeptide chain was shown to be sensitive to hydrolysis by hydroxylamine and also by the widely used serine-protease inhibitor phenylmethylsulfonyl fluoride.

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the oxidative O2-dependent aromatization of the colorless protoporphyrinogen IX to the highly conjugated protoporphyrin IX, the precursor of both hemes and chlorophylls (Fig. 1). It is the final enzyme in the common branch of the heme and chlorophyll biosyn-thetic pathways in plants (Fig. 2).

Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the K m for protoporphyrinogen and the K i for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

Adoptive T-cell therapies, where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition, have shown significant promise in initial clinical trials. However, present adoptive immunotherapy Methods are limited by the need for manipulation of autologous patient T-cells. To permit such an approach in an allogeneic context, Transcription Activator-Like Effector Nucleases (TALENTM) have been used to simultaneously inactivate the endogenous T cell receptor and CD52, a cellular target for a lymphodepleting treatment. This approach reduces the risk of GVHD while permitting proliferation and activity of the introduced T lymphocytes in the presence of the immunosuppressive drug alemtuzumab. Electroporation of primary T cells with mRNA coding for the appropriate TALENTM result in double knock-out (dKO) frequencies of up to 70%. Furthermore, functional characterization demonstrates that the dKO cells are resistant to complement dependent lysis or in vivo depletion by alemtuzumab, and show no apparent potential for TCR-mediated activation. Finally, endowing the dKO cells with a CD19 CAR supports their capacity to kill CD19+ tumor targets as efficiently as unedited T-cells both in vitro and in vivo. Disclosures: Poirot: CELLECTIS THERAPEUTICS: Employment. Schiffer-Mannioui:CELLECTIS THERAPEUTICS: Employment. Philip:UCL Cancer Institute, London, United Kingdom: Employment. Derniame:CELLECTIS THERAPEUTICS: Employment. Gouble:CELLECTIS THERAPEUTICS: Employment. Chion-Sotinel:CELLECTIS THERAPEUTICS: Employment. Le Clerre:CELLECTIS THERAPEUTICS: Employment. Lemaire:CELLECTIS THERAPEUTICS: Employment. Grosse:CELLECTIS THERAPEUTICS: Employment. Cheung:UCL Cancer Institute, London, United Kingdom: Employment. Arnould:CELLECTIS THERAPEUTICS: Employment. Smith:CELLECTIS THERAPEUTICS: Employment. Pule:UCL Cancer Institute, London, United Kingdom: Employment. Scharenberg:CELLECTIS THERAPEUTICS: Employment.

Meeting abstracts Encouraging data have emerged from adoptive T-cell therapies in advanced forms of cancer. Anti-tumor immunity is found in tumor infiltrating lymphocytes as well as engineered T cells where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition on the

Meeting abstracts Recent data have emerged from adoptive T-cell therapies where exogenous expression of a chimeric antigen receptor (CAR) has been shown to confer cancer recognition on autologous T cells. However, the ability to apply this technology in an allogeneic setting would permit the