~150 words and referenced]Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies 1-4 . We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells 5 . Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to 'shuttle' the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for nonviral genome reprogramming that we successfully applied in regulatory T cells
(Tregs), -T cells, B cells, NK cells, and primary and iPS-derived 6 hematopoietic stem progenitor cells (HSPCs).We recently reported an approach to reprogram human T cells with CRISPR-based genome targeting without the need for viral vectors 5 . However, many research and clinical applications still depend upon improved efficiency, cell viability, and generalizability of non-viral genome targeting across cell types 1-4 . We previously found that varying the relative concentrations of both Cas9 RNP and HDR template had significant effects on targeting efficiency
Immunosuppressive activity of conditioned medium from cultured ovine, caprine, and hybrid trophoblast tissue was examined. Conceptuses were obtained from naturally mated donor ewes and does at d 20 of gestation and trophoblast tissue was cultured for 24 h in medium supplemented with 15% calf serum and 1% antibiotic/antimycotic. Conditioned medium was added to pokeweed mitogen-stimulated sheep and goat lymphocyte cultures. Quantification of [3H]thymidine uptake by cells was used to measure lymphocyte proliferation. Ovine, caprine, and hybrid conditioned medium effectively suppressed sheep and goat lymphocyte proliferation (P less than .01). There were no differences (P greater than .05) between the immunosuppressive activity of the three tissue types on either sheep or goat lymphocytes. For all treatment groups, sheep lymphocytes were suppressed more than goat lymphocytes (P less than .05). These results indicate that, at d 20 of gestation, sheep x goat hybrid trophoblast tissue is capable of suppressing pokeweed mitogen-stimulated lymphocyte proliferation.
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