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Meganucleases are important tools for genome engineering, providing an efficient way to generate DNA double-strand breaks at specific loci of interest. Numerous experimental efforts, ranging from in vivo selection to in silico modeling, have been made to re-engineer meganucleases to target relevant DNA sequences. Results: Here we present a novel in silico method for designing custom meganucleases that is based on the use of a machine learning approach. We compared it with existing in silicodoi:10.1186/1471-2105-15-191 pmid:24934562 pmcid:PMC4065607 fatcat:xyl2wjir3ba2xe4l5pk6wvaxqu
more »... ical models and high-throughput experimental screening. The machine learning model was used to successfully predict active meganucleases for 53 new DNA targets. Conclusions: This new method shows competitive performance compared with state-of-the-art in silico physical models, with up to a fourfold increase in terms of the design success rate. Compared to experimental high-throughput screening methods, it reduces the number of screening experiments needed by a factor of more than 100 without affecting final performance.
The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. Adoptive immunotherapy using engineered T-cells hasdoi:10.1038/srep18950 pmid:26750734 pmcid:PMC4707440 fatcat:pqkdu67xjjb2tawbamocbyemgi
more »... erged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering and transfer of chimeric antigen receptors (CARs) or engineered TCRs 1 . Numerous clinical studies have demonstrated the potential of adoptive transfer of CAR T cells for cancer therapy 2-5 but also raised the risks associated with the cytokine-release syndrome (CRS) and the "on-target off-tumor" effect 3,6-8 . To date, few strategies have been developed to pharmacologically control CAR engineered T-cells and may rely on suicide mechanisms 9-14 . Such suicide strategies leading to a complete eradication of the engineered T-cells will result in the premature end of the treatment. Consequently, implementing non-lethal control of engineered CAR T-cells represents an important advancement to improve the CAR T-cell technology and its safety. Small molecule based approaches that rely on dimerizing partner proteins have already been used to study, inter alia, the mechanism of T-cell receptor triggering 15 . Very recently, Lim and colleagues have adapted this approach to control engineered T-cells through the use of a multichain receptor 16 . Here, we describe a strategy to create a switchable engineered CAR T-cells. Our approach is based on engineering a system that is directly integrated in the hinge domain that separate the scFv from the cell membrane. In addition, we chose to implement this strategy in a novel CAR architecture that relies on the FceRI receptor scaffold. The particularity of this design reside in the possibility to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. In this report, we showed that the hinge engineering approaches allowed to turn a T-cell endowed with an engineered CAR from an off-state to an on-state. By controlling the scFv presentation at the cell surface upon addition of the small molecule, our system allowed to further induce the cytolytic properties of the engineered T-cell. Overall, this non-lethal system offers the advantage of a "transient CAR T-cell" for safety while letting open the possibility of multiple specific cytotoxicity cycles using a small molecule drug. Results Experimental setup and CAR architecture. The CAR T-cell performance is intimately linked to an optimal interaction of the scFv to the targeted antigen. We thus conceived a system where controlled of the hinge that separates the scFv from the cell membrane could be obtained upon addition of a small molecule. As a first proof of concept, we focused on the well described and widely used macrolide rapamycin that binds with high affinity to the FKBP12 protein, creating a complex that subsequently interacts with a domain of mTOR (FKBP-rapamycin binding, FRB) 17,18 . In addition, we chose to implement this molecular switch strategy in a novel CAR architecture based on the Fcε RI receptor scaffold, an oligomeric complex composed of three different polypeptide chains (alpha, beta and gamma) 19, 20 . The native activation domains on the gamma and beta subunits were substituted by the intracytoplasmic signaling region of the ζ -chain of the CD3-T cell receptor and by the signaling domains from co-stimulatory 4-1BB (CD137) respectively. The extracellular domain of the alpha subunit was substituted
A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features. We further report a strategy to control TALEN targeting based on the exclusion capacities of ncRVDs (discrimination between differentdoi:10.1038/srep08150 pmid:25632877 pmcid:PMC4311247 fatcat:w2jgo3t6a5hc7j3ov2otg3mzri
more »... otides). By implementing such ncRVDs, we demonstrated in living cells the possibility to efficiently promote TALEN-mediated processing of a target in the HBB locus and alleviate undesired off-site cleavage. We anticipate that this method can greatly benefit to designer nucleases, especially for therapeutic applications and synthetic biology.
Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities. We demonstrate the modularity of the chimeric DmoCre meganuclease previously described, by successfullydoi:10.1093/nar/gkp1171 pmid:20026587 pmcid:PMC2847234 fatcat:r2qrdoljerbx3pdwnymhm2nsqq
more »... ling mutants with locally altered specificities affecting both I-DmoI and I-CreI subdomains in order to create active meganucleases with altered specificities. Moreover these new engineered DmoCre variants appear highly specific and present a low toxicity level, similar to I-SceI, and can induce efficient homologous recombination events in mammalian cells. The DmoCre based meganucleases can therefore offer new possibilities for various genome engineering applications.
Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CARdoi:10.1186/s12896-019-0537-3 pmid:31269942 pmcid:PMC6610870 fatcat:6rfztgykgjgbxei7boupkib6wq
more »... -cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations.
Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders. Previous studies have shown strong crosstalk between differentdoi:10.1093/nar/gkr186 pmid:21482539 pmcid:PMC3152339 fatcat:ahsxovohnzaw7mru3qk2aqbxbu
more »... dues and regions of the DNA binding interface. To investigate this phenomenon, we systematically combined mutations from three groups of amino acids in the DNA binding regions of the I-CreI HE. Our results confirm that important crosstalk occurs throughout this interface in I-CreI. Detailed analysis of success rates identified a nearest-neighbour effect, with a more pronounced level of dependence between adjacent regions. Taken together, these data suggest that combinatorial engineering does not necessarily require the identification of separable functional or structural regions, and that groups of amino acids provide acceptable building blocks that can be assembled, overcoming the context dependency of the DNA binding interface. Furthermore, the present work describes a sequential method to engineer tailored HEs, wherein three contiguous regions are individually mutated and assembled to create HEs with engineered specificity.
The adult neuromuscular junction displays an accumulation of both the acetylcholine receptor (AChR) protein in the subneural domain of the post-synaptic membrane and the mRNAs coding for all its subunits at the level of the subjunctional "fundamental nuclei." In the course of end plate development, the ⑀-subunit, at variance with other subunits, becomes exclusively expressed at the level of the fundamental nuclei, yet at a rather late stage (around birth). To analyze the promoter region of thedoi:10.1074/jbc.271.29.17433 pmid:8663316 fatcat:smjxwz2uvnf47ksau3ykljhcce
more »... -subunit gene which directs its specific expression at the synapse, we used a quantitative transient in vivo expression assay in intact muscle tissue using constructs of the ⑀-subunit promoter placed upstream of the ␤-galactosidase reporter gene. One crucial element for synapse-specific expression was detected between the ؊11 and ؊6 positions. Disruption of this element, either by a scanning mutation or single base mutations, greatly diminishes, or even completely inhibits, preferential expression of the transgene at the end plate. Gel shift experiments reveal the presence of a complex in nuclear muscle extracts that bind the core sequence of this element. The identification of such a site opens the possibility to identify regulatory factors responsible for compartmentalized expression at the neuromuscular junction.
Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRa constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improvedoi:10.1016/j.omtn.2017.10.005 pmid:29246309 pmcid:PMC5684446 fatcat:i7ot47haffhrhknldmrzx2nlwy
more »... ineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.
Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (.14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low.doi:10.1093/nar/gkl720 pmid:17130168 pmcid:PMC1702487 fatcat:mkly3dl3i5hv5hicegh3g4mzze
more »... erefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.
FEBS Open Bio
The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such asdoi:10.1002/2211-5463.13292 pmid:34510816 pmcid:PMC8727936 fatcat:yfuntxskl5euxi6cuvicokqeeq
more »... toxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.
et al.. Comprehensive analysis of the specificity of transcription activator-like effector nucleases. ABSTRACT A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNAtargeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable.doi:10.1093/nar/gku155 pmid:24569350 pmcid:PMC4005648 fatcat:t257klmxgrf3ffaokue3kzqf4e
more »... , we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.
The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases.doi:10.1186/1471-2199-15-13 pmid:24997498 pmcid:PMC4099384 fatcat:7fekoz7kujbypn46zjlvxwc2oi
more »... sults: In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N-and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion: Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.
Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive CAR T-cells scaffold that evades NK cell and alloresponsive T-cell attacks and imparts efficient antitumordoi:10.1101/2021.12.06.471451 fatcat:tasiuffysbfyxjv6kp365lei5m
more »... y in vitro and in vivo. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for future powerful clinical applications.
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