The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR␣), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR␣ on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR␣-blocking antibodies, we demonstrated FR␣-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR␣ was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.
In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).
Gene and cell-based therapies are considered to be potentially powerful new approaches for the management of cystic fibrosis (CF) lung disease. Despite tremendous efforts that have been made, especially in studies to understand the obstacles to gene delivery, major challenges to the application of these approaches remain to be solved. This article will review the advancements made and challenges remaining in the development of viral vector-mediated and cell-based approaches to treat patients with CF.
Transmission of arenaviruses from rodent hosts to humans is generally thought to occur through inhalation or ingestion of dust or droplets containing viral particles. Here we demonstrate that two identified arenavirus receptors, ␣-dystroglycan (␣-DG) and transferrin receptor 1 (TfR1), are expressed in polarized human airway epithelia. Lymphocytic choriomeningitis virus strains with high or low ␣-DG affinity and Junin virus, which binds TfR1, efficiently infected polarized epithelia only when applied to the basolateral surface or when injury compromised tight junction integrity. Viral egress from infected epithelia exhibited basolateral polarity. This study demonstrates that respiratory entry of arenaviruses occurs via basolateral receptors.
Introduction Glecaprevir/pibrentasvir (G/P) was approved on 26 September 2019 by the US Food and Drug Administration for 8-week duration in treatment-naïve (TN) hepatitis C virus (HCV)-infected patients with compensated cirrhosis (CC). Evidence from the EXPEDITION-8 study demonstrated that 8 weeks of G/P achieved a 98% intent-to-treat (ITT) sustained virologic response rate 12 weeks post treatment (SVR12) in 343 TN/CC patients. The aim of this study is to demonstrate the first US real-world effectiveness of G/P 8-week treatment in genotype 1–6 TN/CC HCV patients. Methods Data from 73 TN/CC patients who initiated 8 weeks of G/P treatment between August 2017 and November 2018 were collected electronically from providers and specialty pharmacies of the Trio Health network and analyzed. Cirrhosis was determined by FIB-4 > 5.2 or was physician reported. The primary outcome was Per Protocol (PP) SVR12. Results The majority (60%) of patients were male, with (mean values): age 59 years, body mass index (BMI) of 30, aspartate aminotransferase (AST) 105, and alanine aminotransferase (ALT) 101 IU/ml. HCV genotypes (GT) were: GT1 81% (59/73), GT2 10% (7/73), GT3 5% (4/73), GT4 3% (2/73), and GT6 1% (1/73). Eight percent (6/73) of patients had concurrent proton pump inhibitor (PPI) use, and 15% (11/72) had a baseline viral load > 6 MM IU/ml. Zero patients discontinued, two patients were reported as lost to follow-up, and there was one virologic failure. PP sustained virologic response at 12 weeks (SVR12) rate was 99% (70/71), and the intent-to-treat (ITT) SVR12 rate was 96% (70/73). Conclusions Early real-world experience indicates high effectiveness of the 8-week G/P regimen in a diverse treatment-naïve, compensated cirrhotic US population.
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