Associating growth factor secretions and transcriptomes of single cells in nanovials using SEC-seq.

Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells. Our data indicate that VEGF-A secretion is heterogeneous across the cell population and is poorly correlated with the VEGFA transcript level. The highest VEGF-A secretion occurs in a subpopulation of mesenchymal stromal cells characterized by a unique gene expression signature comprising a surface marker, interleukin-13 receptor subunit alpha 2 (IL13RA2), which allowed the enrichment of this subpopulation. SEC-seq enables the identification of gene signatures linked to specific secretory states, facilitating mechanistic studies, the isolation of secretory subpopulations and the development of means to modulate cellular secretion.

Evaluation of the Elements of Short Hairpin RNAs in Developing shRNA-Containing CAR T Cells.

Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.

Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation.

Human T-lymphotropic virus type I (HTLV-I) infects CD4+ T-cells resulting in a latent, life-long infection in patients. Crosstalk between oncogenic viral factors results in the transformation of the host cell into an aggressive cancer, adult T-cell leukemia/lymphoma (ATL). ATL has a poor prognosis with no currently available effective treatments, urging the development of novel therapeutic strategies. Recent evidence exploring those mechanisms contributing to ATL highlights the viral anti-sense gene HTLV-I bZIP factor (HBZ) as a tumor driver and a potential therapeutic target. In this work, a series of zinc-finger protein (ZFP) repressors were designed to target within the HTLV-I promoter that drives HBZ expression at highly conserved sites covering a wide range of HTLV-I genotypes. ZFPs were identified that potently suppressed HBZ expression and resulted in a significant reduction in the proliferation and viability of a patient-derived ATL cell line with the induction of cell cycle arrest and apoptosis. These data encourage the development of this novel ZFP strategy as a targeted modality to inhibit the molecular driver of ATL, a possible next-generation therapeutic for aggressive HTLV-I associated malignancies.

Conditionally Replicating Vectors Mobilize Chimeric Antigen Receptors against HIV.

Human immunodeficiency virus (HIV) is an attractive target for chimeric antigen receptor (CAR) therapy. CAR T cells have proved remarkably potent in targeted killing of cancer cells, and we surmised that CAR T cells could prove useful in eradicating HIV-infected cells. Toward this goal, we interrogate several neutralizing single-chain variable fragments (scFvs) that target different regions of the HIV envelope glycoprotein, gp120. We find here that CAR T cells with scFv from NIH45-46 antibody demonstrated the highest cytotoxicity. Although NIH45-46 CAR T cells are capable of eliminating antigen-expressing cells, we wanted to address HIV reactivation from ex vivo culture of HIV patient-derived CAR T cells. In order to capitalize on the HIV reactivation, we developed a conditionally replicating lentiviral vector (crLV). The crLV can hijack HIV machinery, forming a chimeric lentivirus (LV) instead of HIV and delivered to uninfected cells. We find that CAR T cells generated with crLVs have similar CAR-mediated functionality as traditional CARs. We also demonstrate crLVs' capability of expanding CAR percentage and protecting CD4 CAR T cell in HIV donors. Collectively, we demonstrate here that the novel crLV NIH45-46 CAR can serve as a strategy to combat HIV, as well as overcome HIV reactivation in CD4+ CAR T cells.