Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.

Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor ß (TGF-ß) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-ß signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.

Non-Modulator Therapies: Developing a Therapy for Every Cystic Fibrosis Patient.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy brings hope to most patients with cystic fibrosis (CF), but not all. For approximately 12% of CF patients with premature termination codon mutations, large deletions, insertions, and frameshifts, the CFTR modulator therapy is not effective. Many believe that genetic-based therapies such as RNA therapies, DNA therapies, and gene editing technologies will be needed to treat mutations that are not responsive to modulator therapy. Delivery of these therapeutic agents to affected cells is the major challenge that will need to be overcome if we are to harness the power of these emerging therapies for the treatment of CF.

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.

Recruitment of monocytes primed to express heme oxygenase-1 ameliorates pathological lung inflammation in cystic fibrosis.

Overwhelming neutrophilic inflammation is a leading cause of lung damage in many pulmonary diseases, including cystic fibrosis (CF). The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway mediates the resolution of inflammation and is defective in CF-affected macrophages (MΦs). Here, we provide evidence that systemic administration of PP-007, a CO releasing/O2 transfer agent, induces the expression of HO-1 in a myeloid differentiation factor 88 (MyD88) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-dependent manner. It also rescues the reduced HO-1 levels in CF-affected cells induced in response to lipopolysaccharides (LPS) or Pseudomonas aeruginosa (PA). Treatment of CF and muco-obstructive lung disease mouse models with a single clinically relevant dose of PP-007 leads to effective resolution of lung neutrophilia and to decreased levels of proinflammatory cytokines in response to LPS. Using HO-1 conditional knockout mice, we show that the beneficial effect of PP-007 is due to the priming of circulating monocytes trafficking to the lungs in response to infection to express high levels of HO-1. Finally, we show that PP-007 does not compromise the clearance of PA in the setting of chronic airway infection. Overall, we reveal the mechanism of action of PP-007 responsible for the immunomodulatory function observed in clinical trials for a wide range of diseases and demonstrate the potential use of PP-007 in controlling neutrophilic pulmonary inflammation by promoting the expression of HO-1 in monocytes/macrophages.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

BACKGROUND: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of ß-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. METHODS AND FINDINGS: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. CONCLUSIONS: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

Understanding the Host in the Management of Pneumonia. An Official American Thoracic Society Workshop Report.

Pneumonia causes a significant burden of disease worldwide. Although all populations are at risk of pneumonia, those at extremes of age and those with immunosuppressive disorders, underlying respiratory disease, and critical illness are particularly vulnerable. Although clinical practice guidelines addressing the management and treatment of pneumonia exist, few of the supporting studies focus on the crucial contributions of the host in pneumonia pathogenesis and recovery. Such essential considerations include the host risk factors that lead to susceptibility to lung infections; biomarkers reflecting the host response and the means to pursue host-directed pneumonia therapy; systemic effects of pneumonia on the host; and long-term health outcomes after pneumonia. To address these gaps, the Pneumonia Working Group of the Assembly on Pulmonary Infection and Tuberculosis led a workshop held at the American Thoracic Society meeting in May 2018 with overarching objectives to foster attention, stimulate research, and promote funding for short-term and long-term investigations into the host contributions to pneumonia. The workshop involved participants from various disciplines with expertise in lung infection, pneumonia, sepsis, immunocompromised patients, translational biology, data science, genomics, systems biology, and clinical trials. This workshop report summarizes the presentations and discussions and important recommendations for future clinical pneumonia studies. These recommendations include establishing consensus disease and outcome definitions, improved phenotyping, development of clinical study networks, standardized data and biospecimen collection and protocols, and development of innovative trial designs.

Nanoparticles for delivery of agents to fetal lungs.

Fetal treatment of congenital lung disease, such as cystic fibrosis, surfactant protein syndromes, and congenital diaphragmatic hernia, has been made possible by improvements in prenatal diagnostic and interventional technology. Delivery of therapeutic agents to fetal lungs in nanoparticles improves cellular uptake. The efficacy and safety of nanoparticle-based fetal lung therapy depends on targeting of necessary cell populations. This study aimed to determine the relative distribution of nanoparticles of a variety of compositions and sizes in the lungs of fetal mice delivered through intravenous and intra-amniotic routes. Intravenous delivery of particles was more effective than intra-amniotic delivery for epithelial, endothelial and hematopoietic cells in the fetal lung. The most effective targeting of lung tissue was with 250nm Poly-Amine-co-Ester (PACE) particles accumulating in 50% and 44% of epithelial and endothelial cells. This study demonstrated that route of delivery and particle composition impacts relative cellular uptake in fetal lung, which will inform future studies in particle-based fetal therapy.

Emerging technologies for cystic fibrosis transmembrane conductance regulator restoration in all people with CF.

Although effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has the potential to change the lives of many patients with cystic fibrosis (CF), it is unlikely that these drugs will be a game changing therapy for all. There are about 10% of patients with CF who don't produce a mutant protein tomodulate, potentiate, or optimize and for these patients such therapies are unlikely to be of significant benefit. There is a need to develop new therapeutic approaches that can work for this patient population and can advance CF therapies. These new therapies will be genetic-based therapies and each approach will result in functional CFTR protein inpreviously affected CF cells. In this review we will examine the potential of RNA therapies, gene transfer therapies, and gene editing therapies for the treatment of CF as well as the challenges that will need to be facedas we harness the power of these emerging therapies towards a one-time cure.

Single-Cell Transcriptional Archetypes of Airway Inflammation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is a life-shortening, multisystem hereditary disease caused by abnormal chloride transport. CF lung disease is driven by innate immune dysfunction and exaggerated inflammatory responses that contribute to tissue injury. To define the transcriptional profile of this airway immune dysfunction, we performed the first single-cell transcriptome characterization of CF sputum.Objectives: To define the transcriptional profile of sputum cells and its implication in the pathogenesis of immune function and the development of CF lung disease.Methods: We performed single-cell RNA sequencing of sputum cells from nine subjects with CF and five healthy control subjects. We applied novel computational approaches to define expression-based cell function and maturity profiles, herein called transcriptional archetypes.Measurements and Main Results: The airway immune cell repertoire shifted from alveolar macrophages in healthy control subjects to a predominance of recruited monocytes and neutrophils in CF. Recruited lung mononuclear phagocytes were abundant in CF and were separated into the following three archetypes: activated monocytes, monocyte-derived macrophages, and heat shock-activated monocytes. Neutrophils were the most prevalent in CF, with a dominant immature proinflammatory archetype. Although CF monocytes exhibited proinflammatory features, both monocytes and neutrophils showed transcriptional evidence of abnormal phagocytic and cell-survival programs.Conclusions: Our findings offer an opportunity to understand subject-specific immune dysfunction and its contribution to divergent clinical courses in CF. As we progress toward personalized applications of therapeutic and genomic developments, we hope this inflammation-profiling approach will enable further discoveries that change the natural history of CF lung disease.

Cystic fibrosis transmembrane conductance receptor modulator therapy in cystic fibrosis, an update.

PURPOSE OF REVIEW: Cystic fibrosis transmembrane conductance receptor (CFTR) modulators are a new class of drugs that treat the underlying cause of cystic fibrosis. To date, there are four approved medications, which are mutation-specific. Although the number of mutations that respond to these agents is expanding, effective CFTR modulators are not available to all cystic fibrosis patients. The purpose of this article is to review the approved CFTR modulators and discuss the mutations that can be treated with these agents, as well as, review the long-term benefits of modulator therapy. RECENT FINDINGS: More people with cystic fibrosis can be effectively treated with CFTR modulators. The new, highly effective triple therapy, elexacaftor/tezacaftor/ivacaftor is indicated for more than 90% of patients with cystic fibrosis and ivacaftor is now approved for children as young as 6 months of age with 1 of 30 CFTR mutations. Long-term use of modulator therapy is associated with fewer pulmonary exacerbations, maintenance of lung function, improved weight gain, and quality of life. SUMMARY: CFTR modulators are the first therapies developed to treat the underlying defect in cystic fibrosis. Their use is associated with preserved lung function and improved health in patients with cystic fibrosis.

Ezrin links CFTR to TLR4 signaling to orchestrate anti-bacterial immune response in macrophages.

Macrophages (MΦs) with mutations in cystic fibrosis transmembrane conductance regulator (CFTR) have blunted induction of PI3K/AKT signaling in response to TLR4 activation, leading to hyperinflammation, a hallmark of cystic fibrosis (CF) disease. Here, we show that Ezrin links CFTR and TLR4 signaling, and is necessary for PI3K/AKT signaling induction in response to MΦ activation. Because PI3K/AKT signaling is critical for immune regulation, Ezrin-deficient MΦs are hyperinflammatory and have impaired Pseudomonas aeruginosa phagocytosis, phenocopying CF MΦs. Importantly, we show that activated CF MΦs have reduced protein levels and altered localization of the remaining Ezrin to filopodia that form during activation. In summary, we have described a direct link from CFTR to Ezrin to PI3K/AKT signaling that is disrupted in CF, and thus promotes hyper-inflammation and weakens phagocytosis.

Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion.

Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr-/- mice in Ussing chambers and measured transcellular secretion of [14C]oxalate. Intestinal tissue isolated from Cftr-/- mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr-/- tissue. Compared with wild-type mice, Cftr-/- mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl--oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr-/- mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis.

Increased susceptibility of Cftr-/- mice to LPS-induced lung remodeling.

Cystic fibrosis (CF) is caused by homozygous mutations of the CF transmembrane conductance regulator (CFTR) Cl(-) channel, which result in chronic pulmonary infection and inflammation, the major cause of morbidity and mortality. Although these processes are clearly related to each other, each is likely to contribute to the pathology differently. Understanding the contribution of each of these processes to the overall pathology has been difficult, because they are usually so intimately connected. Various CF mouse models have demonstrated abnormal immune responses compared with wild-type (WT) littermates when challenged with live bacteria or bacterial products acutely. However, these studies have not investigated the consequences of persistent inflammation on lung tissue in CF mice, which may better model the lung pathology in patients. We characterized the lung pathology and immune response of Cftr(-/-) (CF) and Cftr(+/+) (WT) mice to chronic administration of Pseudomonas aeruginosa lipopolysaccharide (LPS). We show that, after long-term repeated LPS exposure, CF mice develop an abnormal and persistent immune response, which is associated with more robust structural changes in the lung than those observed in WT mice. Although CF mice and their WT littermates develop lung pathology after chronic exposure to LPS, the inflammation and damage resolve in WT mice. However, CF mice do not recover efficiently, and, as a consequence of their chronic inflammation, CF mice are more susceptible to morphological changes and lung remodeling. This study shows that chronic inflammation alone contributes significantly to aspects of CF lung pathology.

Genetics of Cystic Fibrosis: Clinical Implications.

Cystic fibrosis (CF) is a common life-shortening autosomal recessive genetic disorder caused by mutations in the gene that encodes for the cystic fibrosis transmembrane conductance regulator protein (CFTR). Almost 2000 variants in the CFTR gene have been identified. The mutational classes are based on the functional consequences on CFTR. New therapies are being developed to target mutant CFTR and restore CFTR function. Understanding specific CF genotypes is essential for providing state-of-the art care to patients. In addition to the variation in CFTR genotype, there are several modifier genes that contribute to the respiratory phenotype.

Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI.

Myosin Ia (Myo1a), the most prominent plus-end directed motor and myosin VI (Myo6) the sole minus-end directed motor, together exert opposing tension between the microvillar (MV) actin core and the apical brush border (BB) membrane of the intestinal epithelial cell (IEC). Mice lacking Myo1a or Myo6 each exhibit a variety of defects in the tethering of the BB membrane to the actin cytoskeleton. Double mutant (DM) mice lacking both myosins revealed that all the defects observed in either the Myo1a KO or Snell's waltzer (sv/sv) Myo6 mutant mouse are absent. In isolated DM BBs, Myo1a crosslinks between MV membrane and MV actin core are absent but the gap (which is lost in Myo1a KO) between the MV core and membrane is maintained. Several myosins including Myo1c, d, and e and Myo5a are ectopically recruited to the BB. Consistent with the restoration of membrane tethering defects by one or more of these myosins, upward ATP-driven shedding of the BB membrane, which is blocked in the Myo1a KO, is restored in the DM BB. However, Myo1a or Myo6 dependent defects in expression of membrane proteins that traffic between the BB membrane and endosome (NaPi2b, NHE3, CFTR) are not restored. Compared to controls, Myo1a KO, sv/sv mice exhibit moderate and DM high levels of hypersensitivity to dextran sulfate sodium-induced colitis. Consistent with Myo1a and Myo6 playing critical roles in maintaining IEC integrity and response to injury, DM IECs exhibit increased numbers of apoptotic nuclei, above that reported for Myo1a KO.

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

Association between serum 25-hydroxyvitamin D level and pulmonary exacerbations in cystic fibrosis.

OBJECTIVE: To identify effects of vitamin D status, as defined by circulating 25-hydroxyvitamin D (25-OHD) levels on the annual frequency of pulmonary exacerbations (Pex) and hospitalizations, on standard measures of pulmonary function, and to identify determinants of 25-OHD levels in pediatric patients with cystic fibrosis (CF). HYPOTHESIS: Higher levels of serum 25-OHD would be associated with fewer Pex and hospitalizations, and improved lung function based on pulmonary function tests (PFTs). STUDY DESIGN: Retrospective chart review of 53 pediatric patients from January 2011 to December 2011. Significant relationships were examined using linear and logistic regression analyses. PATIENT SELECTION: Patients ages 5 through 22 years followed at the CF Care Center and Clinic at Yale-New Haven Hospital, New Haven, CT., who had at least four clinic visits and at least one 25-OHD measurement between January 1, 2011 and December 31, 2011. RESULTS: Serum 25-OHD level and gender were strong independent determinants of the annual number of Pex (P < 0.01, with lower 25-OHD level and female gender associated with Pex). There was a significant influence of gender (P < 0.05) and a near-significant influence of serum 25-OHD (P < 0.08) on hospitalization rate. There was no effect of 25-OHD levels on PFTs. Other candidate factors (age, season, gender, pancreatic sufficiency, or severity of genetic mutation) did not significantly effect 25-OHD level. CONCLUSIONS: The annual number of Pex in pediatric CF patients is significantly associated with 25-OHD levels and gender, raising the consideration that maintaining vitamin D sufficiency may lead to decreased incidence of Pex and hospitalizations requiring antibiotic therapy.

Pharmacological modulation of the AKT/microRNA-199a-5p/CAV1 pathway ameliorates cystic fibrosis lung hyper-inflammation.

In cystic fibrosis (CF) patients, hyper-inflammation is a key factor in lung destruction and disease morbidity. We have previously demonstrated that macrophages drive the lung hyper-inflammatory response to LPS in CF mice, because of reduced levels of the scaffold protein CAV1 with subsequent uncontrolled TLR4 signalling. Here we show that reduced CAV1 and, consequently, increased TLR4 signalling, in human and murine CF macrophages and murine CF lungs, is caused by high microRNA-199a-5p levels, which are PI3K/AKT-dependent. Downregulation of microRNA-199a-5p or increased AKT signalling restores CAV1 expression and reduces hyper-inflammation in CF macrophages. Importantly, the FDA-approved drug celecoxib re-establishes the AKT/miR-199a-5p/CAV1 axis in CF macrophages, and ameliorates lung hyper-inflammation in Cftr-deficient mice. Thus, we identify the AKT/miR-199a-5p/CAV1 pathway as a regulator of innate immunity, which is dysfunctional in CF macrophages contributing to lung hyper-inflammation. In addition, we found that this pathway can be targeted by celecoxib.