No abstract

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Xeroderma pigmentosum group C (XP-C) is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG) located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases, Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells. Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.

We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy.

Double-strand break (DSB)-induced homologous recombination (HR) of direct repeats is a powerful means to achieve gene excision, a critical step in genome engineering. In this report we have used an extrachrmosomal reporter system to monitor the impact of different parameters on meganuclease-induced HR in CHO-K1 cells. We found that repeat homology length is critical. Virtually no HR could be detected with a 15-bp duplication, while, with repeats larger than 400 bp, recombination efficiency became less dependent on homology length. The presence of an intervening sequence between the duplications dramatically impairs HR, independent of the cleavage position; by 3 kb of insertion, HR is virtually undetectable. Efficient HR can be restored by positioning cleavage sites at both ends of the intervening sequence, allowing a constant level of excision with up to 10 kb of intervening sequences. Using similar constructs, 2.8-kb inserts could be efficiently removed from several chromosomal loci, illustrating the wide potential of this technology. These results fit current models of direct repeat recombination and identify DSB-induced HR as a powerful tool for gene excision.

Background: Engineered meganucleases are one of most promising biological reagents for gene modification therapy. Results: CpG methylation affects engineered meganuclease activity and DNA binding affinity in a position-dependent manner. Conclusion:The activity and sequence specificity of engineered meganucleases are not the only parameters to consider for successful gene edition. Significance: Considering epigenetic factors is crucial for designing highly active engineered meganucleases for gene editing purposes.

Glioblastoma multiforme (GBM) is a highly aggressive form of brain tumors with a 5-year survival rate of less than 10%. Standard-of-care combining radiation therapy with temozolomide only yields a median survival of 14.6 months and more effective therapeutic options to extend patient lives are urgently needed. EGFR variant III (EGFRvIII) is a tumor specific mutant of EGFR found in 25-30% of GBM but not expressed in healthy tissues. EGFRvIII is formed by an in-frame deletion of exons 2-7 of the wild-type EGFR which leads to removal of 267 amino acids in the extracellular domain of the receptor. The truncated receptor loses its ability to bind ligands but acquires constitutive kinase activity. The lack of normal tissue expression makes EGFRvIII an ideal target for developing CAR T therapy. Most CAR T therapies in clinical development use the patients’ own T cells for CAR T manufacturing. Our approach is to develop an “off-the-shelf” CAR-T treatment for GBM using healthy donor T cells with the TCR (T-cell receptor) disrupted to prevent graft-versus-host disease. This allogeneic approach could circumvent challenges faced by some patients due to limited availability and quality of their own T cells and rapid progress of their diseases, and has the potential to reduce the high cost associated with autologous CART therapy. Using phage panning and hybridoma approaches, we generated a series of humanized and fully human EGFRvIII antibodies with a wide range of affinities. Recombinant EGFRvIII protein binding by Biacore and FACS assays using EGFRvIII and EGFR wild-type expressing cells demonstrated that these antibodies are highly specific for EGFRvIII. The antibodies were converted to ScFvs and cloned into a CAR vector. Subsequently, EGFRvIII CARs were transduced into primary human T cells for functional studies. We developed a series of assays to evaluate CAR expression, degranulation activity and target dependent cytotoxicity. Our goal is to select CAR candidate that is safe, persistent and has potent target dependent cytotoxicity. Citation Format: Oi Kwan Wong, Mathilde Dusseaux, Jing-Tyan Ma, Melinda Au, Sophie Leduc, Joyce Chou, Jessica M. Yu, Marjorie Bateman, Thomas Pertel, Kevin C. Lindquist, Julianne Smith, Barbra Sasu, Shu-Hui Liu. Allogeneic EGFRvIII chimeric antigen receptor T cells for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3751. doi:10.1158/1538-7445.AM2017-3751

Sickle cell disease (SCD) is one of the most common inherited diseases affecting millions of people worldwide. SCD stems from a single point mutation (A>T) in exon 1 of the HBB gene which results in sickle hemoglobin. The only available curative treatment of SCD is allogeneic hematopoietic stem cell transplant, which is only viable for ~20% of SCD patients. Ex-vivo gene therapy approaches have shown to be a promising therapeutic option for patients. Most products currently in the clinic have focused on methods to enhance functional hemoglobin production (e.g., disruption of BCL11A to encourage β-globin gene switching or direct insertion of a functional β-globin gene into patient HSPCs). The Cellectis approach is to directly repair the mutated HBB gene in order to restore HbA production. TALGlobin01 is an autologous HSPC-based gene therapy product designed with a TALEN ® optimized to cleave the sickle HBB gene (TALEN-HBB01) and an AAV based engineering process leading to highly efficient HBB gene correction via endogenous homology directed repair (HDR), while mitigating potential risks of HBB gene knock-out (KO). Use of TALGlobin01 resulted in up to 70% of HDR-mediated HBB gene correction (56% mean frequency) in homozygous sickle (HbSS) patient HSPCs with only 20% of NHEJ-dependent insertion/deletion (indels) events detected. This gene correction process did not affect cell viability, hematopoietic stem/progenitor immunophenotype or differentiation potential of corrected HSPCs. Allelic editing at clonal resolution in single BFU-E colonies showed that up to 72% (with a mean of 50%) of progenitors contained at least one corrected allele, while only 23% were either not corrected or had indels on one allele. Notably, our optimized engineering process led to only 9% of colonies harboring bi-allelic indels events. To evaluate the ability of TALGlobin01 to prevent the sickling phenotype associated with SCD, we performed in vitro differentiation of HbSS patient-edited cells into late-stage erythroid cells and assessed HbA protein production by HPLC. We observed that HbA accounted for up to 60% (with a mean of 49%) of the total Hb with a concomitant decrease of HbS production from 90% to 19%. Interestingly, our gene correction process maintained a balanced α chain/non-α chain ratio, consistent with our genotyping results showing a low frequency of clones harboring bi-allelic indels. More importantly, efficient expression of HbA was translated into a sharp decrease of hypoxia-induced sickling rate of in vitro-generated erythrocytes when comparing unedited to TALGlobin01 edited cells (from 95% to 13%, respectively). When injected in vivo, engineered HSPCs from non-mobilized SCD patients retained the capacity to engraft into immunodeficient NSG mice with levels of allelic correction comparable to the input (~40%) and still detectable at 16-17 weeks post-transplantation. An unbiased genome wide approach (OCA), coupled to target enrichment high-throughput sequencing screening, confirmed the TALEN-HBB01 cleavage activity at only one off-target site located at the HBD locus. TALEN-HBB01 cleavage activity was assessed at this off-target site in patient HSPCs engineered with TALGlobin01 and found to be very low compared to the on-site cleavage activity (50.7% indels at the on-site versus 1.2% at the off-site). Furthermore, we developed a clinical-scale TALGlobin01 manufacturing process that achieved up to 60% of HDR-mediated HBB gene correction and less than 10% of NHEJ-mediated HBB gene inactivation in healthy donor derived HSPCs. These results are the first demonstration that a TALEN-based engineering process could be used to efficiently correct the SCD mutated HBB gene in HbSS patient-derived CD34+ HSPCs. Taken together, the high level of HbA expression and reversion of sickling phenotype, the efficient in vivo long-term engraftment potential of TALGlobin01 edited cells and the low levels of HBB KO or off-target cleavage generated by our gene correction process, warrant the clinical evaluation of TALGlobin01 to treat SCD patients. Disclosures Moiani: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Hong: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Letort: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Lizot: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Chirinos: Cellectis, S.A.: Current Employment. Temburni-Blake: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Mayer: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Leduc: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Pinard: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Foray: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Boyne: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Kazancioglu: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Dusseaux: Cellectis, S.A.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Gouble: Cellectis SA: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Frattini: Celgene/BMS: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Brownstein: BMS: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Duclert: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Shiffer-Mannoui: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Juillerat: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Duchateau: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Valton: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.