James R. Rose

Efficient delivery of nucleic acids to the lung has the potential to impact future respiratory treatments. Current formulations exhibit significant limitations, including insufficient potency, single‐cell delivery characterizations, efficacy demonstrations in disease models, and toxicology assessments. Here we describe polymer 81 (P81), which, when used with hypotonic solvents, is 34.1‐ to 77.8‐fold more potent than previous polymers with various cargos for lung transfection by nebulized mRNA. Through the exploration of multiple routes of administration, P81 demonstrated a wide of range of cell‐type delivery, including bronchial airway epithelial cells, tracheal basal stem cells, and 54% of all cells in the distal lung, assessed via single‐cell spatial transcriptomics. Robust expression of mRNA‐encoded antibodies in the lung by P81 enabled quantification of target protein and successful suppression of SARS‐CoV‐2 infection. The toxicology assessments of multiple doses of P81 polyplexes via nebulization in rodents were unremarkable. Overall, P81 represents a step forward for inhaled nucleic acid delivery to the respiratory tract, positioning itself as an efficient, safe, and translatable delivery formulation.

Image-based spatial transcriptomics depends on cell segmentation to assign transcripts to individual cells, but how segmentation algorithms perform across tissues with distinct cellular architectures is poorly understood. This study presents the broadest independent benchmark to date of cell segmentation algorithms for spatial transcriptomics, comparing five approaches across ten mouse tissues using a 5,006-gene Xenium panel. To quantify segmentation errors, Co-expression Rejection in Segmentation Purity (CRISP) was developed, an open-source tool available in R and Python that measures cell purity through tissue-specific mutually exclusive marker co-expression without requiring ground truth annotations. This benchmark revealed that segmentation algorithms face a fundamental tradeoff between maximizing transcript capture and maintaining cell purity, and that the severity of this tradeoff is tissue-dependent. Proseg achieved the highest average performance across tissues, though the magnitude of its advantage varies with tissue architecture. Overall, CRISP provides per-tissue performance profiles as a practical resource for algorithm selection.

Cell division is an essential component of B cell differentiation to Ab-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell–specific deletion of IRF4. IRF4-deficient B cells responding to influenza, 4-hydroxy-3-nitrophenylacetyl–Ficoll, and LPS divided but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient cells was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4–MYC–mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.