The function of a T cell depends on its subtype and activation state. Here, we show that the imaging of autofluorescence-lifetime signals from quiescent and activated T cells can be used to Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model.ResultsIn comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.ConclusionsThis study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed “living drugs” for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.

Regulatory Factor X (RFX) transcription factors (TFs) are best known for activating genes required for ciliogenesis in both vertebrates and invertebrates. In humans, eight RFX TFs have a variety of tissue-specific functions, while in the worm Caenorhabditis elegans, the sole RFX gene, daf-19, encodes a set of nested isoforms. Null alleles of daf-19 confer pleiotropic effects including altered development with a dauer constitutive phenotype, complete absence of cilia and ciliary proteins, and defects in synaptic protein maintenance. We sought to identify RFX/daf-19 target genes associated with neuronal functions other than ciliogenesis using comparative transcriptome analyses at different life stages of the worm. Subsequent characterization of gene expression patterns revealed one set of genes activated in the presence of DAF-19 in ciliated sensory neurons, whose activation requires the daf-19c isoform, also required for ciliogenesis. A second set of genes is downregulated in the presence of DAF-19, primarily in nonsensory neurons. The human orthologs of some of these neuronal genes are associated with human diseases. We report the novel finding that daf-19a is directly or indirectly responsible for downregulation of these neuronal genes in C. elegans by characterizing a new mutation affecting the daf-19a isoform (tm5562) and not associated with ciliogenesis, but which confers synaptic and behavioral defects. Thus, we have identified a new regulatory role for RFX TFs in the nervous system. The new daf-19 candidate target genes we have identified by transcriptomics will serve to uncover the molecular underpinnings of the pleiotropic effects that daf-19 exerts on nervous system function.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

New gene editing tools like CRISPR-Cas9 enable precision genome engineering within cell lines, primary cells, and model organisms, with some formulations now entering the clinic. “Precision” applies to various aspects of gene editing, and can be tailored for each application. Here we review recent advances in four types of precision in gene editing: 1) increased DNA cutting precision (e.g., on-target:off-target nuclease specificity), 2) increased on-target knock-in of sequence variants and transgenes (e.g., increased homology-directed repair), 3) increased transcriptional control of edited genes, and 4) increased specificity in delivery to a specific cell or tissue. Design of next-generation gene and cell therapies will likely exploit a combination of these advances.

24Dominantly inherited disorders are not typically considered therapeutic candidates for gene 25 augmentation (GA). We tested whether GA or genome editing (GE) could serve as a solo therapy 26 for autosomal dominant Best disease (adBD), a macular dystrophy linked to over 100 mutations in 27 the BEST1 gene, which encodes a homo-pentameric calcium-activated chloride channel (CaCC) in 28 the retinal pigment epithelium (RPE). Since no suitable animal models of adBD exist, we generated 29 RPE from patient-derived induced pluripotent stem cells (iPSC-RPE) and found that GA restored 30 CaCC activity and improved rhodopsin degradation in a subset of adBD lines. iPSC-RPE 31 harboring adBD mutations in calcium clasp or chloride binding domains of the channel, but not in 32 a putative structural region, were responsive to GA. However, reversal of the iPSC-RPE CaCC 33 deficit was demonstrated in every adBD line following targeted CRISPR-Cas9 GE of the mutant 34 allele. Importantly, 95% of GE events resulted in premature stop codons within the mutant allele, 35 and single cell profiling demonstrated no adverse perturbation of RPE transcriptional programs 36 post-editing. These results show that GA is a viable approach for a subset of adBD patients 37 depending on the functional role of the mutated residue. Further, GA non-responders are 38 candidates for targeted GE of the mutant allele. Similar scenarios likely exist for other 39 genotypically diverse dominant diseases, expanding the therapeutic landscape for affected patients. 40 41Genotypically heterogeneous dominant diseases pose significant challenges and opportunities for 43 precision medicine (Doudna and Charpentier, 2014). Among gene therapies, GA for recessive disorders is 44 the most developed, having spurred multiple clinical trials (Cukras et al., 2018; Lam et al., 2019; Russell 45 et al., 2017) and FDA approval for one ocular disease (Ledford, 2017). However, GA is generally ruled 46 out as a stand-alone therapy for dominant disorders due to a perceived need to eliminate the deleterious 47 WT BEST1 was elucidated, which revealed it to be a homo-pentameric CaCC (Dickson et al., 2014; Yang 64 et al., 2014) (Figure 1A). Mutation hotspots in BEST1 were found to lie within calcium or chloride ion 65 binding sites, or contribute to the structural organization of the channel, among other roles (Dickson et al., 66 2014). Our two prior adBD iPSC-RPE models harbored mutations in a calcium binding (N296H) or 67 structural (A146K) domain (Singh et al., 2015;Singh et al., 2013b). Therefore, for the present study, we 68 generated iPSCs from a third patient with adBD caused by a chloride binding site mutation (R218C), as 69 well as an ARB patient with compound heterozygous mutations (R141H/A195V) (Figure 1B,C). We also 70 4 employed two control lines: a WT iPSC line and an isogenic line generated via CRISPR-based gene 71 correction of R218C adBD iPSCs (Steyer et al., 2018). 72 73 RESULTS 74BEST1 protein is robustly expressed in WT and adBD iPSC-RPE, but not ARB iPSC...