Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney.

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.