A roadmap to precision treatments for familial pulmonary fibrosis.

Interstitial lung diseases (ILDs) in adults and children (chILD) are a heterogeneous group of lung disorders leading to inflammation, abnormal tissue repair and scarring of the lung parenchyma often resulting in respiratory failure and death. Inherited factors directly cause, or contribute significantly to the risk of developing ILD, so called familial pulmonary fibrosis (FPF), and monogenic forms may have a poor prognosis and respond poorly to current treatments. Specific, variant-targeted or precision treatments are lacking. Clinical trials of repurposed drugs, anti-fibrotic medications and specific treatments are emerging but for many patients no interventions exist. We convened an expert working group to develop an overarching framework to address the existing research gaps in basic, translational, and clinical research and identified areas for future development of preclinical models, candidate medications and innovative clinical trials. In this Position Paper, we summarise working group discussions, recommendations, and unresolved questions concerning precision treatments for FPF.

Beyond a team: The comprehensive interdisciplinary endocarditis program in the United States.

Although multidisciplinary teams have been shown to decrease in-hospital mortality for patient with infectious endocarditis, most studies have focused on the inpatient role of these teams, and are primarily based at European tertiary care centers. There is limited literature available on the optimal longitudinal care of this patient population. Here we outline our experience developing an interdisciplinary endocarditis program at the University of Kentucky, which cares for patients from their index hospitalization into the outpatient setting, while also coordinating transfers from regional hospitals and offering education to regional providers.

Workplace-based assessments-Articulating the playbook.

INTRODUCTION: A workplace-based assessment (WBA) is a learning recording device that is widely used in medical education globally. Although entrenched in medical curricula, and despite a substantial body of literature exploring them, it is not yet fully understood how WBAs play out in practice. Adopting a constructivist standpoint, we examine these assessments, in the workplace, using principles based upon naturalist inquiry, drawing from a theoretical framework based on Goffman's dramaturgical analogy for the presentation of self, and using qualitative research methods to articulate what is happening as learners complete them. METHODS: Learners were voluntarily recruited to participate in the study from a single teaching hospital. Data were generated, in-situ, through observations with field notes and audiovisual recording of WBAs, along with accompanying interviews with learners. RESULTS: Data from six learners was analysed to reveal a set of general principles-the WBA playbook. These four principles were tacit, unwritten, unofficial and learners applied them to complete their WBA proformas: (1) maintain the impression of progression, (2) manage the authenticity of the individual proforma, (3) avoid losing face with the assessor and (4) complete the proforma in an effort-efficient way. By adhering to these principles, learners expressed their understanding of their social position in their world at that time the documents were created. DISCUSSION: This paper recognises the value of the WBA as a lived experience, and of the WBA document as a social space, where learners engage in a social performance before the readers of the proforma. Such an interpretation better represents what happens as learners undergo and record WBAs in the real-world, recognising WBAs as learner-centred, learner-driven, meaning-making phenomena. In this way, as a record of interpretation and meanings, the subjective nature of the WBA process is a strength to be harnessed, rather than a weakness to be glossed over.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

Type I interferon receptor signalling deficiency results in dysregulated innate immune responses to SARS-CoV-2 in mice.

SARS-CoV-2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID-19). The type I interferon (IFN) pathway is of particular importance for anti-viral defense and recent studies identified that type I IFNs drive early inflammatory responses to SARS-CoV-2. Here, we use a mouse model of SARS-CoV-2 infection, facilitating viral entry by intranasal recombinant Adeno-Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN receptor-1 deficient (Ifnar1-/- ) mice, to study the role of type I IFN signalling and innate immune responses during SARS-CoV-2 infection. Our data show that type I IFN signalling is essential for inducing anti-viral effector responses to SARS-CoV-2, control of virus replication, and to prevent enhanced disease. Furthermore, hACE2-Ifnar1-/- mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as reduced and delayed production of monocyte-recruiting chemokine CCL2. hACE2-Ifnar1-/- mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS-CoV-2 infection, with a shift from Ly6C+ to Ly6C- expressing cells. Together, our findings suggest that type I IFN signalling deficiency results in a dysregulated innate immune response to SARS-CoV-2 infection.

Sendai F/HN pseudotyped lentiviral vector transduces human ciliated and non-ciliated airway cells using α 2,3 sialylated receptors.

A lentiviral vector (LV) pseudotype derived from the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of a murine respirovirus (Sendai virus) facilitates efficient targeting of murine lung in vivo. Since targeting of the human lung will depend upon the availability and distribution of receptors used by F/HN, we investigated transduction of primary human airway cells differentiated at the air-liquid interface (ALI). We observed targeting of human basal, ciliated, goblet, and club cells, and using a combination of sialidase enzymes and lectins, we showed that transduction is dependent on the availability of sialylated glycans, including α2,3 sialylated N-acetyllactosamine (LacNAc). Transduction via F/HN was 300-fold more efficient than another hemagglutinin-based LV pseudotype derived from influenza fowl plague virus (HA Rostock), despite similar efficiency reported in murine airways in vivo. Using specific glycans to inhibit hemagglutination, we showed this could be due to a greater affinity of F/HN for α2,3 sialylated LacNAc. Overall, these results highlight the importance of identifying the receptors used in animal and cell-culture models to predict performance in the human airways. Given the reported prevalence of α2,3 sialylated LacNAc on human pulmonary cells, these results support the suitability of the F/HN pseudotype for human lung gene therapy applications.

Correction of a chronic pulmonary disease through lentiviral vector-mediated protein expression.

We developed a novel lentiviral vector, pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN), that produces long-lasting, high-efficiency transduction of the respiratory epithelium. Here we addressed whether this platform technology can secrete sufficient levels of a therapeutic protein into the lungs to ameliorate a fatal pulmonary disease as an example of its translational capability. Pulmonary alveolar proteinosis (PAP) results from alveolar granulocyte-macrophage colony-stimulating factor (GM-CSF) insufficiency, resulting in abnormal surfactant homeostasis and consequent ventilatory problems. Lungs of GM-CSF knockout mice were transduced with a single dose of rSIV.F/HN-expressing murine GM-CSF (mGM-CSF; 1e5-92e7 transduction units [TU]/mouse); mGM-CSF expression was dose related and persisted for at least 11 months. PAP disease biomarkers were rapidly and persistently corrected, but we noted a narrow toxicity/efficacy window. rSIV.F/HN may be a useful platform technology to deliver therapeutic proteins for lung diseases requiring long-lasting and stable expression of secreted proteins.

Intranasal Lentiviral Vector-Mediated Antibody Delivery Confers Reduction of SARS-CoV-2 Infection in Elderly and Immunocompromised Mice.

Vaccines for COVID-19 are now a crucial public health need, but the degree of protection provided by conventional vaccinations for individuals with compromised immune systems is unclear. The use of viral vectors to express neutralizing monoclonal antibodies (mAbs) in the lung is an alternative approach that does not wholly depend on individuals having intact immune systems and responses. Here, we identified an anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibody, NC0321, which can efficiently neutralize a range of SARS-CoV-2 variants, including alpha, beta, delta, and eta. Both prophylactic and therapeutic NC0321 treatments effectively protected mice from SARS-CoV-2 infection. Notably, we adopted viral vector-mediated delivery of NC0321 IgG1 as an attractive approach to prevent SARS-CoV-2 infection. The NC0321 IgG1 expression in the proximal airway, expressed by a single direct in-vivo intranasal (I.N.) administration of a self-inactivating and recombinant lentiviral vector (rSIV.F/HN-NC0321), can protect young, elderly, and immunocompromised mice against mouse-adapted SARS-CoV-2 surrogate challenge. Long-term monitoring indicated that rSIV.F/HN-NC0321 mediated robust IgG expression throughout the airway of young and SCID mice, importantly, no statistical difference in the NC0321 expression between young and SCID mice was observed. A single I.N. dose of rSIV.F/HN-NC0321 30 or 180 days prior to SARS-CoV-2 challenge significantly reduced lung SARS-CoV-2 titers in an Ad5-hACE2-transduced mouse model, reconfirming that this vectored immunoprophylaxis strategy could be useful, especially for those individuals who cannot gain effective immunity from existing vaccines, and could potentially prevent clinical sequelae.

Lung directed antibody gene transfer confers protection against SARS-CoV-2 infection.

BACKGROUND: The COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. AIMS: We hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. METHODS: We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. CONCLUSIONS: A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.

RNA-seq analysis of the human surfactant air-liquid interface culture reveals alveolar type II cell-like transcriptome.

Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured in vitro long term and, although engineered organoids are an attractive choice, they do not phenotypically recapitulate the lung parenchyma; overall, these models do not allow for the generation of reliable disease models. Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model.

Sharing is caring: an analysis of #FOAMed Twitter posts during the COVID-19 pandemic.

PURPOSE: Free Open Access Medical Education (FOAMed) is a worldwide social media movement designed to accelerate and democratise the sharing of medical knowledge. This study sought to investigate the content shared through FOAMed during the emerging COVID-19 pandemic. STUDY DESIGN: Tweets containing the #FOAMed hashtag posted during a 24-hour period in April 2020 were studied. Included tweets were analysed using the Wiig knowledge management cycle framework (building knowledge, holding knowledge, pooling knowledge and using knowledge). RESULTS: 1379 tweets contained the #FOAMed hashtag, of which 265 met the inclusion criteria and were included in the analysis. Included tweets were posted from 208 distinct users, originated from each world continent and were in five different languages. Three overarching themes were identified: (1) signposting and appraising evidence and guidelines; (2) sharing specialist and technical advice; and (3) personal and social engagement. Among 12 subthemes within these groupings, 11 aligned to one of the four dimensions of the Wiig knowledge management cycle framework, and the other focused on building and managing social networks. Almost 40% of tweets related directly to COVID-19. CONCLUSION: #FOAMed tweets during the COVID-19 pandemic included a broad range of resources, advice and support. Despite the geographical, language and disciplinary variation of contributing users and the lack of organisational structure uniting them, this social media medical community has been able to construct, share and use emerging technical knowledge through a time of extraordinary challenge and uncertainty for the global medical community.

Breaking Borders: How Barriers to Global Mobility Hinder International Partnerships in Academic Medicine.

This article describes the authors' personal experiences of collaborating across international borders in academic research. International collaboration in academic medicine is one of the most important ways by which research and innovation develop globally. However, the intersections among colonialism, academic medicine, and global health research have created a neocolonial narrative that perpetuates inequalities in global health partnerships. The authors critically examine the visa process as an example of a racist practice to show how the challenges of blocked mobility increase inequality and thwart research endeavors. Visas are used to limit mobility across certain borders, and this limitation hinders international collaborations in academic medicine. The authors discuss the concept of social closure and how limits to global mobility for scholars from low- and middle-income countries perpetuate a cycle of dependence on scholars who have virtually barrier-free global mobility-these scholars being mainly from high-income countries. Given the current sociopolitical milieu of increasing border controls and fears of illegal immigration, the authors' experiences expose what is at stake for academic medicine when the political sphere, focused on tightening border security, and the medical realm, striving to build international research collaborations, intersect. Creating more equitable global partnerships in research requires a shift from the current paradigm that dominates most international partnerships and causes injury to African scholars.

Identification of LZTFL1 as a candidate effector gene at a COVID-19 risk locus.

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target.

Genomic diversity of SARS-CoV-2 in Oxford during United Kingdom's first national lockdown.

Epidemiological efforts to model the spread of SARS-CoV-2, the virus that causes COVID-19, are crucial to understanding and containing current and future outbreaks and to inform public health responses. Mutations that occur in viral genomes can alter virulence during outbreaks by increasing infection rates and helping the virus evade the host immune system. To understand the changes in viral genomic diversity and molecular epidemiology in Oxford during the first wave of infections in the United Kingdom, we analyzed 563 clinical SARS-CoV-2 samples via whole-genome sequencing using Nanopore MinION sequencing. Large-scale surveillance efforts during viral epidemics are likely to be confounded by the number of independent introductions of the viral strains into a region. To avoid such issues and better understand the selection-based changes occurring in the SARS-CoV-2 genome, we utilized local isolates collected during the UK's first national lockdown whereby personal interactions, international and national travel were considerably restricted and controlled. We were able to track the short-term evolution of the virus, detect the emergence of several mutations of concern or interest, and capture the viral diversity of the region. Overall, these results demonstrate genomic pathogen surveillance efforts have considerable utility in controlling the local spread of the virus.

Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection.

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.

A human surfactant B deficiency air-liquid interface cell culture model suitable for gene therapy applications.

Surfactant protein B (SPB) deficiency is a severe monogenic interstitial lung disorder that leads to loss of life in infants as a result of alveolar collapse and respiratory distress syndrome. The development and assessment of curative therapies for the deficiency are limited by the general lack of well-characterized and physiologically relevant in vitro models of human lung parenchyma. Here, we describe a new human surfactant air-liquid interface (SALI) culture model based on H441 cells, which successfully recapitulates the key characteristics of human alveolar cells in primary culture as evidenced by RNA and protein expression of alveolar cell markers. SALI cultures were able to develop stratified cellular layers with functional barrier properties that are stable for at least 28 days after air-lift. A SFTPB knockout model of SPB deficiency was generated via gene editing of SALI cultures. The SFTPB-edited SALI cultures lost expression of SPB completely and showed weaker functional barrier properties. We were able to correct this phenotype via delivery of a lentiviral vector pseudotyped with Sendai virus glycoproteins F/HN expressing SPB. We believe that SALI cultures can serve as an important in vitro research tool to study human alveolar epithelium, especially for the development of advanced therapy medicinal products targeting monogenic disorders.

Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal's lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids.

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.

Lung-targeting lentiviral vector for passive immunisation against influenza.

When recombinant simian immunodeficiency virus (SIV) is pseudotyped with the F and HN glycoproteins from murine respiratory Sendai virus (rSIV.F/HN), it provides efficient lung cell targeting and lifelong transgene expression in the murine airways. We have shown that a single dose of rSIV.F/HN can direct stable expression of neutralising antibody against influenza in the murine airways and systemic circulation, and protects mice against two different influenza strains in lethal challenge experiments. These data suggest that rSIV.F/HN could be used as a vector for passive immunisation against influenza and other respiratory pathogens.