Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with diverse disorders and characterized by disruption of the alveolar-capillary barrier, leakage of edema fluid into the lung, and substantial inflammation leading to acute respiratory failure. Gene therapy is a potentially powerful approach to treat ALI/ARDS through repair of alveolar epithelial function. Herein, we show that delivery of a plasmid expressing β1-subunit of the Na(+),K(+)-ATPase (β1-Na(+),K(+)-ATPase) alone or in combination with epithelial sodium channel (ENaC) α1-subunit using electroporation not only protected from subsequent lipopolysaccharide (LPS)-mediated lung injury, but also treated injured lungs. However, transfer of α1-subunit of ENaC (α1-ENaC) alone only provided protection benefit rather than treatment benefit although alveolar fluid clearance had been remarkably enhanced. Gene transfer of β1-Na(+),K(+)-ATPase, but not α1-ENaC, not only enhanced expression of tight junction protein zona occludins-1 (ZO-1) and occludin both in cultured cells and in mouse lungs, but also reduced pre-existing increase of lung permeability in vivo. These results demonstrate that gene transfer of β1-Na(+),K(+)-ATPase upregulates tight junction formation and therefore treats lungs with existing injury, whereas delivery of α1-ENaC only maintains pre-existing tight junction but not for generation. This indicates that the restoration of epithelial/endothelial barrier function may provide better treatment of ALI/ARDS.
Introduction Acute Respiratory Distress Syndrome (ARDS) is a common cause of organ failure with an associated mortality rate of 40%. The initiating event is disruption of alveolar-capillary interface causing leakage of edema into alveoli. Hypothesis: Electroporation mediated gene delivery of epithelial sodium channel (ENaC) and Na+,K+-ATPase into alveolar cells would improve alveolar clearance of edema and attenuate ARDS. Methods Pigs were anesthetized, instrumented, and the superior mesenteric artery was clamped to cause gut ischemia/reperfusion injury (I/R) and peritoneal sepsis (PS) by fecal clot implantation. Animals were ventilated according to ARDSnet protocol. Four hours after injury animals were randomized into groups: 1.Treatment Na+,K+-ATPase/ENaC plasmid (n=5), 2.Control Empty plasmid (n=5). Plasmids were delivered to the lung using bronchoscope. Electroporation was delivered using 8 square wave electric pulses across chest. Following electroporation pigs were monitored 48 hrs. Results The PaO2/FiO2 ratio and lung compliance were higher in the treatment group. Lung Wet/Dry Ratio was lower in the treatment group. Relative expression of the Na+,K+-ATPase transgene was higher throughout lungs receiving treatment plasmids. Quantitative histopathology revealed a reduction in intra-alveolar fibrin in the Treatment group. Bronchoalveolar lavage showed increased surfactant protein B in the treatment group. Survival was improved in the treatment group. Conclusion Electroporation-mediated transfer of Na+,K+-ATPase/ENaC plasmids improved lung function, reduced fibrin deposits, decreased lung edema, and improved survival in a translational porcine model of ARDS. Gene therapy can attenuate ARDS pathophysiology in a high fidelity animal model suggesting a potential new therapy for patients.
Zika virus (ZiKV) is a mosquito-borne member of theFlaviviridae family that has been known to circulate for decades causing mild febrile illness. the more recent ZiKV outbreaks in the Americas and the caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. in this study we demonstrate that direct delivery in mice of an infectious ZiKV cDnA clone allows the rescue of recombinant (r)ZiKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. the rZiKV recovered from these mice remained fully virulent in a second passage in mice. interestingly, infectious rZiKV was also recovered after intraperitoneal inoculation of the rZiKV cDnA in the absence of transfection reagent. further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDnA clone encoding an attenuated rZiKV was safe, highly immunogenic, and provided full protection against lethal ZiKV challenge. this novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and liveattenuated ZiKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.Zika virus (ZIKV), a member of the Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly 1 . The viral genome is made of a positive sense, single-stranded RNA molecule (~10.8 kb) that contains a single open reading frame flanked by 5′ and 3′ untranslated regions 2-4 . The viral RNA is translated as a single polyprotein that is co-and post-translationally processed by viral and cellular proteases into three major structural proteins (capsid, pre-membrane and envelope) involved in viral entry, fusion and assembly 5 , and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) that are involved in viral RNA replication and transcription, assembly and evasion of the host antiviral responses [6][7][8][9] .Although the majority of ZIKV infections are asymptomatic or associated with a mild febrile illness, infection during pregnancy has been associated with miscarriage and severe congenital malformations, including fetal
The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, especially in the lung. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. We demonstrate, through microinjection of fluorescently labeled plasmids, that a DNA sequence within the rat T1α promoter was able to mediate ATI cell-specific plasmid DNA nuclear import due to the binding of ATI-enriched transcription factors. Promoter deletion analysis and site-directed mutagenesis of specific transcription-factor-binding sites within the +101 to -200 bp region of the T1α promoter identified HNF3 and TTF-1 as critical transcription factors for import. To test for nuclear import in vivo, plasmids expressing GFP from the CMV promoter were delivered into the lungs of mice by electroporation and evaluated immunohistochemically 48 h later. Plasmids carrying the 1.3 kbp T1α sequence resulted in GFP expression almost exclusively in ATI cells. This represents a new and highly efficient way to target a specific lung epithelial cell type both in vitro and in vivo based on the restriction of DNA nuclear import.
Idiopathic pulmonary fibrosis (IPF) is a devastating and fatal disease and characterized by increased deposition of extracellular matrix proteins and scar formation in the lung, resulting from alveolar epithelial damage and accumulation of inflammatory cells. Evidence suggests that Caveolin-1 (Cav-1), a major component of caveolae which regulates cell signaling and endocytosis, is a potential target to treat fibrotic diseases, although the mechanisms and responsible cell types are unclear. We show that Cav-1 expression was downregulated both in alveolar epithelial type I cells in bleomycin-injured mouse lungs and in lung sections from IPF patients. Increased expression of IL-1β and caspase-1 has been observed in IPF patients, indicating inflammasome activation associated with IPF. Gene transfer of a plasmid expressing Cav-1 using transthoracic electroporation reduced infiltration of neutrophils and monocytes/macrophages and protected from subsequent bleomycin-induced pulmonary fibrosis. Overexpression of Cav-1 suppressed bleomycin- or silica-induced activation of caspase-1 and maturation of pro-IL-1β to secrete cleaved IL-1β both in mouse lungs and in primary type I cells. These results demonstrate that gene transfer of Cav-1 downregulates inflammasome activity and protects from subsequent bleomycin-mediated pulmonary fibrosis. This indicates a pivotal regulation of Cav-1 in inflammasome activity and suggests a novel therapeutic strategy for patients with IPF.
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