CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages.

An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5' end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.

Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery.

The low yield of extracellular vesicle (EV) secretion is a major obstacle for mass production and limits their potential for clinical applications as a drug delivery platform. Here, we mass produced engineered extracellular vesicles (eEVs) by fusing the surface composition of EVs with lipid-based materials via a membrane extrusion technique. A library of lipids (DOTAP, POPC, DPPC and POPG) was fused with EVs to form a hybrid-lipid membrane structure. Uniform lamellar vesicles with a controlled size around 100 nm were obtained in this study. Particle number characterization revealed this extrusion method allowed a 6- to 43-fold increase in numbers of vesicles post- isolation. Further, exogenous siRNA was successfully loaded into engineered vesicles with ~ 15% - 20% encapsulation efficiency using electroporation technique. These engineered extracellular vesicles sustained a 14-fold higher cellular uptake to lung cancer cells (A549) and achieved an effective gene silencing effect comparable to commercial Lipofectamine RNAiMax. Our results demonstrate the surface composition and functionality of EVs can be tuned by extrusion with lipids and suggest the engineered vesicles can be a potential substitute as gene delivery carriers while being able to be mass produced to a greater degree with retained targeting capabilities of EVs.

Controlling and quantifying the stability of amino acid-based cargo within polymeric delivery systems.

In recent years, the rapid growth and availability of protein and peptide therapeutics has not only expanded the boundaries of modern science but has also revolutionized the practice of medicine today. The potential of such therapies, however, is greatly limited by the innate instabilities of proteins and peptides, which is further magnified during therapeutic formulation processing, transport, storage, and administration. In this paper, we will consider the unique stability challenges associated with protein/peptide polymeric delivery systems from an engineering approach oriented towards the quantification and modification of amino acid-based cargo stability. While a number of methods have been developed for the purposes of quantifying factors affecting protein and peptide stability, current measurement techniques remain largely limited in scope in regard to polymeric drug delivery systems. This paper will primarily describe the influence of water content, pH, and temperature on protein and peptide stability within polymer-based delivery systems. Moreover, we will review current instrumentation used to quantify factors affecting protein/peptide stability with respect to water content, pH, and temperature. Lastly, we will outline several recommendations to help guide future research efforts to develop methods more specific to quantifying protein/peptide stability within polymer-based delivery systems.