Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that in vivo genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.

Single-cell RNA-Sequencing has the potential to provide deep biological insights by revealing complex regulatory interactions across diverse cell phenotypes at single-cell resolution. However, current single-cell gene regulatory network inference methods produce a single regulatory network per input dataset, limiting their capability to uncover complex regulatory relationships across related cell phenotypes. We present SimiC, a single-cell gene regulatory inference framework that overcomes this limitation by jointly inferring distinct, but related, gene regulatory dynamics per phenotype. We show that SimiC uncovers key regulatory dynamics missed by previously proposed methods across a range of systems, both model and non-model alike. In particular, SimiC was able to uncover CAR T cell dynamics after tumor recognition and key regulatory patterns on a regenerating liver, and was able to implicate glial cells in the generation of distinct behavioral states in honeybees. SimiC hence establishes a new approach to quantitating regulatory architectures between distinct cellular phenotypes, with far-reaching implications for systems biology.