Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

RATIONALE AND OBJECTIVE: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. METHODS: Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. RESULTS: rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. CONCLUSIONS: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Spatial transcriptomic characterization of pathologic niches in IPF.

Despite advancements in antifibrotic therapy, idiopathic pulmonary fibrosis (IPF) remains a medical condition with unmet needs. Single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of IPF but lacks the cellular tissue context and gene expression localization that spatial transcriptomics provides. To bridge this gap, we profiled IPF and control patient lung tissue using spatial transcriptomics, integrating the data with an IPF scRNA-seq atlas. We identified three disease-associated niches with unique cellular compositions and localizations. These include a fibrotic niche, consisting of myofibroblasts and aberrant basaloid cells, located around airways and adjacent to an airway macrophage niche in the lumen, containing SPP1+ macrophages. In addition, we identified an immune niche, characterized by distinct lymphoid cell foci in fibrotic tissue, surrounded by remodeled endothelial vessels. This spatial characterization of IPF niches will facilitate the identification of drug targets that disrupt disease-driving niches and aid in the development of disease relevant in vitro models.

Multidrug resistance plasmids commonly reprogram the expression of metabolic genes in Escherichia coli.

Multidrug-resistant Escherichia coli is a leading cause of global mortality. Transfer of plasmids carrying genes encoding beta-lactamases, carbapenamases, and colistin resistance between lineages is driving the rising rates of hard-to-treat nosocomial and community infections. Multidrug resistance (MDR) plasmid acquisition commonly causes transcriptional disruption, and while a number of studies have shown strain-specific fitness and transcriptional effects of an MDR plasmid across diverse bacterial lineages, fewer studies have compared the impacts of different MDR plasmids in a common bacterial host. As such, our ability to predict which MDR plasmids are the most likely to be maintained and spread in bacterial populations is limited. Here, we introduced eight diverse MDR plasmids encoding resistances against a range of clinically important antibiotics into E. coli K-12 MG1655 and measured their fitness costs and transcriptional impacts. The scale of the transcriptional responses varied substantially between plasmids, ranging from >650 to <20 chromosomal genes being differentially expressed. However, the scale of regulatory disruption did not correlate significantly with the magnitude of the plasmid fitness cost, which also varied between plasmids. The identities of differentially expressed genes differed between transconjugants, although the expression of certain metabolic genes and functions were convergently affected by multiple plasmids, including the downregulation of genes involved in L-methionine transport and metabolism. Our data show the complexity of the interaction between host genetic background and plasmid genetic background in determining the impact of MDR plasmid acquisition on E. coli. IMPORTANCE: The increase in infections that are resistant to multiple classes of antibiotics, including those isolates that carry carbapenamases, beta-lactamases, and colistin resistance genes, is of global concern. Many of these resistances are spread by conjugative plasmids. Understanding more about how an isolate responds to an incoming plasmid that encodes antibiotic resistance will provide information that could be used to predict the emergence of MDR lineages. Here, the identification of metabolic networks as being particularly sensitive to incoming plasmids suggests the possible targets for reducing plasmid transfer.

Three dimensional fibrotic extracellular matrix directs microenvironment fiber remodeling by fibroblasts.

Idiopathic pulmonary fibrosis (IPF), for which effective treatments are limited, results in excessive and disorganized deposition of aberrant extracellular matrix (ECM). An altered ECM microenvironment is postulated to contribute to disease progression through inducing profibrotic behavior of lung fibroblasts, the main producers and regulators of ECM. Here, we examined this hypothesis in a 3D in vitro model system by growing primary human lung fibroblasts in ECM-derived hydrogels from non-fibrotic (control) or IPF lung tissue. Using this model, we compared how control and IPF lung-derived fibroblasts responded in control and fibrotic microenvironments in a combinatorial manner. Culture of fibroblasts in fibrotic hydrogels did not alter in the overall amount of collagen or glycosaminoglycans but did cause a drastic change in fiber organization compared to culture in control hydrogels. High-density collagen percentage was increased by control fibroblasts in IPF hydrogels at day 7, but decreased at day 14. In contrast, IPF fibroblasts only decreased the high-density collagen percentage at day 14, which was accompanied by enhanced fiber alignment in IPF hydrogels. Similarly, stiffness of fibrotic hydrogels was increased only by control fibroblasts by day 14 while those of control hydrogels were not altered by fibroblasts. These data highlight how the ECM-remodeling responses of fibroblasts are influenced by the origin of both the cells and the ECM. Moreover, by showing how the 3D microenvironment plays a crucial role in directing cells, our study paves the way in guiding future investigations examining fibrotic processes with respect to ECM remodeling responses of fibroblasts. STATEMENT OF SIGNIFICANCE: In this study, we investigated the influence of the altered extracellular matrix (ECM) in Idiopathic Pulmonary Fibrosis (IPF), using a 3D in vitro model system composed of ECM-derived hydrogels from both IPF and control lungs, seeded with human IPF and control lung fibroblasts. While our results indicated that fibrotic microenvironment did not change the overall collagen or glycosaminoglycan content, it resulted in a dramatically alteration of fiber organization and mechanical properties. Control fibroblasts responded differently from IPF fibroblasts, highlighting the unique instructive role of the fibrotic ECM and the interplay with fibroblast origin. These results underscore the importance of 3D microenvironments in guiding pro-fibrotic responses, offering potential insights for future IPF therapies as well as other fibrotic diseases and cancer.