Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, ..."AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.
•Recent advances in the understanding of murine and human thymic and peripheral Treg development are discussed.•Transcriptional and epigenetic mechanisms regulating Treg differentiation and lineage ...stability are summarized.•Approaches to enhance Tregs, their advantages and limitations are discussed.•Potential utility of these approaches to treat autoimmunity are critically reviewed.
Self-tolerance, the state of unresponsiveness to self-tissues/antigens, is maintained through central and peripheral tolerance mechanisms, and a breach of these mechanisms leads to autoimmune diseases. Foxp3 + T-regulatory cells (Tregs) play an essential role in suppressing autoimmune response directed against self-antigens and thereby regulate self-tolerance. Natural Tregs are differentiated in the thymus on the basis of their higher TCR-affinity to self-antigens and migrate to the periphery where they maintain peripheral tolerance. In addition, extra-thymic differentiation of induced Tregs can occur in the periphery which can control abrupt immune responses under inflammatory conditions. A defect in Treg cell numbers and/or function is found to be associated with the development of autoimmune disease in several experimental models and human autoimmune diseases. Moreover, augmentation of Tregs has been shown to be beneficial in treating autoimmunity in preclinical models, and Treg based cellular therapy has shown initial promise in clinical trials. However, emerging studies have identified an unstable subpopulation of Tregs which expresses pro-inflammatory cytokines under both homeostatic and autoimmune conditions, as well as in ex vivo cultures. In addition, clinical translation of Treg cellular therapy is impeded by limitations such as lack of easier methods for selective expansion of Tregs and higher cost associated with GMP-facilities required for cell sorting, ex vivo expansion and infusion of ex vivo expanded Tregs. Here, we discuss the recent advances in molecular mechanisms regulating Treg differentiation, Foxp3 expression and lineage stability, the role of Tregs in the prevention of various autoimmune diseases, and critically review their clinical utility for treating human autoimmune diseases.
Foxp3
T regulatory cells (Tregs) control autoimmune response by suppressing proliferation and effector functions of self-reactive Foxp3
CD4
/CD8
T cells and thereby maintain the critical balance ...between self-tolerance and autoimmunity. Earlier, we had shown that OX40L-JAG1 cosignaling mediated through their cognate receptors OX40 and Notch3 preferentially expressed on murine Tregs can selectively induce their proliferation in the absence of TCR stimulation. However, the differential molecular mechanisms regulating TCR-independent versus TCR-dependent Treg proliferation and lineage stability of the expanded Tregs remained unknown. In this study, we show that OX40L-JAG1 treatment induced TCR-independent proliferation of Tregs in the thymus and periphery. The use of Src kinase inhibitor permitted us to demonstrate selective inhibition of TCR-dependent T cell proliferation with little to no effect on OX40L-JAG1-induced TCR-independent Treg expansion in vitro, which was critically dependent on noncanonical NF-κB signaling. OX40L-JAG1-expanded Tregs showed sustained lineage stability as indicated by stable demethylation marks in Treg signature genes such as
and
Furthermore, OX40L-JAG1 treatment significantly increased CTLA4
and TIGIT
Tregs and alleviated experimental autoimmune thyroiditis in mice. Relevance of our findings to humans became apparent when human OX40L and JAG1 induced TCR-independent selective expansion of human Tregs in thymocyte cultures and increased human Tregs in the liver tissue of humanized NSG mice. Our findings suggest that OX40L-JAG1-induced TCR-independent Treg proliferation is a conserved mechanism that can be used to expand lineage-stable Tregs to treat autoimmune diseases.
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a ...cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
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