Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Upregulation of alveolar fluid clearance is not sufficient for Na+,K+-ATPase ß subunit-mediated gene therapy of LPS-induced acute lung injury in mice.

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by diffuse alveolar damage and significant edema accumulation, which is associated with impaired alveolar fluid clearance (AFC) and alveolar-capillary barrier disruption, leading to acute respiratory failure. Our previous data showed that electroporation-mediated gene delivery of the Na+, K+-ATPase ß1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS-induced ALI in mice. More importantly, our recent publication showed that gene delivery of MRCKα, the downstream effector of ß1 subunit-mediated signaling towards upregulation of adhesive junctions and epithelial and endothelial barrier integrity, also provided therapeutic potential for ARDS treatment in vivo but without necessarily accelerating AFC, indicating that for ARDS treatment, improving alveolar capillary barrier function may be of more benefit than improving fluid clearance. In the present study, we investigated the therapeutical potential of ß2 and ß3 subunits, the other two ß isoforms of Na+, K+-ATPase, for LPS-induced ALI. We found that gene transfer of either the ß1, ß2, or ß3 subunits significantly increased AFC compared to the basal level in naïve animals and each gave similar increased AFC to each other. However, unlike that of the ß1 subunit, gene transfer of the ß2 or ß3 subunit into pre-injured animal lungs failed to show the beneficial effects of attenuated histological damage, neutrophil infiltration, overall lung edema, or increased lung permeability, indicating that ß2 or ß3 gene delivery could not treat LPS induced lung injury. Further, while ß1 gene transfer increased levels of key tight junction proteins in the lungs of injured mice, that of either the ß2 or ß3 subunit had no effect on levels of tight junction proteins. Taken together, this strongly suggests that restoration of alveolar-capillary barrier function alone may be of equal or even more benefit than improving AFC for ALI/ARDS treatment.

Multiple conductance states of lipid pores during Voltage-Clamp electroporation.

We used voltage-clamp electroporation to obtain single-channel recordings of lipid pores and analyzed the idealized dwell-time sequences using Maximum-Likelihood Fitting. We observed traces with multiple current levels and determined whether they were a result of the presence of multiple pores or a single pore with multiple conductance states. We found that, within the same recording, the bilayer can have a single pore with multiple conductance states or multiple independent pores. Using high sampling rates (100 kHz) we were able to observe pores with 40 µs lifetimes, and in experiments using high-voltage pulses we observed the existence of long-lived fluctuations minutes after the removal of the electric field. These results come closer to reconciling the nanosecond lifetime pores in molecular dynamics simulations and the long-lived permeability of cells after electroporation.

Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury.

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, the administration of Syk inhibitor R788 (fostamatinib); currently in phase II clinical trial for the treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

Modeling and simulation of current-clamp electroporation.

Current-Clamp electroporation refers to the application of a constant current across a membrane which results in voltage fluctuations due to the creation of electropores. This method allows for the measurement of electroporation across a long timescale (minutes) and facilitates the comparison between experimental and theoretical studies. Of particular interest is the claim in the literature that current-clamp electroporation results in the creation of a single pore. We simulated current-clamp electroporation using the Smoluchowski and Langevin equations and identified two possible mechanisms to explain the observed voltage fluctuations. The voltage fluctuations may be due to a single pore or a few pores growing and shrinking via a negative feedback mechanism or the opening and closing of pores in a larger population of pores. Our results suggest that current-clamp conditions do not necessarily result in the creation of a single pore. Additionally, we showed that the Langevin model is more accurate than the Smoluchowski model under conditions where there are only a few pores.

Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function.

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by alveolar edema accumulation with reduced alveolar fluid clearance (AFC), alveolar-capillary barrier disruption, and substantial inflammation, all leading to acute respiratory failure. Enhancing AFC has long been considered one of the primary therapeutic goals in gene therapy treatments for ARDS. We previously showed that electroporation-mediated gene delivery of the Na+, K+-ATPase ß1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS-induced ALI in mice. We identified MRCKα as an interaction partner of ß1 which mediates this upregulation in cultured alveolar epithelial cells. In this study, we investigate whether electroporation-mediated gene transfer of MRCKα to the lungs can attenuate LPS-induced acute lung injury in vivo. Compared to mice that received a non-expressing plasmid, those receiving the MRCKα plasmid showed attenuated LPS-increased pulmonary edema and lung leakage, restored tight junction protein expression, and improved overall outcomes. Interestingly, gene transfer of MRCKα did not alter AFC rates. Studies using both cultured microvascular endothelial cells and mice suggest that ß1 and MRCKα upregulate junctional complexes in both alveolar epithelial and capillary endothelial cells, and that one or both barriers may be positively affected by our approach. Our data support a model of treatment for ALI/ARDS in which improvement of alveolar-capillary barrier function alone may be of more benefit than improvement of alveolar fluid clearance.

Changes in lung immune cell infiltrates after electric field treatment in mice.

Exogenous electric fields are currently used in human therapy in a number of contexts. Interestingly, electric fields have also been shown to alter migration and function of immune cells, suggesting the potential for electric field-based immune therapy. Little is known as to the effect of electric field treatment (EFT) on the lung. To determine if EFT associates with changes in lung immune cell infiltration, we used a mouse model with varying methods of EFT application and measured cells and soluble mediators using flow cytometry and cytokine/chemokine multiplex. EFT was associated with a transient increase in lung neutrophils and decrease in eosinophils in naïve mice within 2 h of treatment, accompanied by an increase in IL-6 levels. In order to test whether EFT could alter eosinophil/neutrophil recruitment in a relevant disease model, a mouse model of allergic airway inflammation was used. Four EFT doses in allergen-sensitized mice resulted in increased neutrophil and reduced eosinophil infiltrates following allergen challenge, suggesting a durable change in inflammation by EFT. Mice with allergic inflammation were analyzed by flexiVent for measures of lung function. EFT-treated mice had increased inspiratory capacity and other measures of lung function were not diminished. These data suggest EFT may be used to manipulate immune cell infiltration in the lung without affecting lung function.

The Na+, K+-ATPase ß1 subunit regulates epithelial tight junctions via MRCKα.

An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA ß1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport-independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the ß1 subunit, including a top candidate, myotonic dystrophy kinase-related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA ß1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.

Gene Therapy for Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome that leads to acute respiratory failure and accounts for over 70,000 deaths per year in the United States alone, even prior to the COVID-19 pandemic. While its molecular details have been teased apart and its pathophysiology largely established over the past 30 years, relatively few pharmacological advances in treatment have been made based on this knowledge. Indeed, mortality remains very close to what it was 30 years ago. As an alternative to traditional pharmacological approaches, gene therapy offers a highly controlled and targeted strategy to treat the disease at the molecular level. Although there is no single gene or combination of genes responsible for ARDS, there are a number of genes that can be targeted for upregulation or downregulation that could alleviate many of the symptoms and address the underlying mechanisms of this syndrome. This review will focus on the pathophysiology of ARDS and how gene therapy has been used for prevention and treatment. Strategies for gene delivery to the lung, such as barriers encountered during gene transfer, specific classes of genes that have been targeted, and the outcomes of these approaches on ARDS pathogenesis and resolution will be discussed.

Synthesis and Application of Peptide-siRNA Nanoparticles from Disulfide-Constrained Cyclic Amphipathic Peptides for the Functional Delivery of Therapeutic Oligonucleotides to the Lung.

The potential of RNAi therapies has been largely impeded by the inherent challenges in the functional delivery of siRNA to cells. Herein, we describe protocols for the synthesis and characterization of novel peptide-siRNA nanoparticles prepared from disulfide-constrained amphipathic peptides complexed with siRNA as promising siRNA delivery vectors. We also describe protocols for the application of these nanoparticles to the in vitro and in vivo delivery of siRNA to lung cells for the functional knockdown of lung proteins.

Pulmonary gene delivery-Realities and possibilities.

Delivery of genetic material to tissues in vivo is an important technique used in research settings and is the foundation upon which clinical gene therapy is built. The lung is a prime target for gene delivery due to a host of genetic, acquired, and infectious diseases that manifest themselves there, resulting in many pathologies. However, the in vivo delivery of genetic material to the lung remains a practical problem clinically and is considered the major obstacle needed to be overcome for gene therapy. Currently there are four main strategies for in vivo gene delivery to the lung: viral vectors, liposomes, nanoparticles, and electroporation. Viral delivery uses several different genetically modified viruses that enter the cell and express desired genes that have been inserted to the viral genome. Liposomes use combinations of charged and neutral lipids that can encapsulate genetic cargo and enter cells through endogenous mechanisms, thereby delivering their cargoes. Nanoparticles are defined by their size (typically less than 100 nm) and are made up of many different classes of building blocks, including biological and synthetic polymers, cell penetrant and other peptides, and dendrimers, that also enter cells through endogenous mechanisms. Electroporation uses mild to moderate electrical pulses to create pores in the cell membrane through which delivered genetic material can enter a cell. An emerging fifth category, exosomes and extracellular vesicles, may have advantages of both viral and non-viral approaches. These extracellular vesicles bud from cellular membranes containing receptors and ligands that may aid cell targeting and which can be loaded with genetic material for efficient transfer. Each of these vectors can be used for different gene delivery applications based on mechanisms of action, side-effects, and other factors, and their use in the lung and possible clinical considerations is the primary focus of this review.

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae 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 live-attenuated 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.

Disruption of normal patterns of FOXF1 expression in a lethal disorder of lung development.

BACKGROUND: Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor FOXF1, which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood. The objective of our study is to gain insights into the mechanisms by which CNVs contribute to an ACDMPV phenotype. METHODS: We analysed primary lung tissue from an infant with classic clinical and histological findings of ACDMPV and harboured a 340 kb deletion on chromosome 16q24.1 located 250 kb upstream of FOXF1. RESULTS: In RNA generated from paraffin-fixed lung sections, our patient had lower expression of FOXF1 than age-matched controls. He also had an abnormal pattern of FOXF1 protein expression, with a dramatic loss of FOXF1 expression in the lung. To gain insights into the mechanisms underlying these changes, we assessed the epigenetic landscape using chromatin immunoprecipitation, which demonstrated loss of histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark of active enhancers, in the region of the deletion. CONCLUSIONS: Together, these data suggest that the deletion disrupts an enhancer responsible for directing FOXF1 expression in the developing lung and provide novel insights into the mechanisms underlying a fatal developmental lung disorder.

Caveolin-1 gene therapy inhibits inflammasome activation to protect from bleomycin-induced pulmonary fibrosis.

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-1ß 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-1ß to secrete cleaved IL-1ß 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.

RNAi therapeutic strategies for acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS), replacing the clinical term acute lung injury, involves serious pathophysiological lung changes that arise from a variety of pulmonary and nonpulmonary injuries and currently has no pharmacological therapeutics. RNA interference (RNAi) has the potential to generate therapeutic effects that would increase patient survival rates from this condition. It is the purpose of this review to discuss potential targets in treating ARDS with RNAi strategies, as well as to outline the challenges of oligonucleotide delivery to the lung and tactics to circumvent these delivery barriers.

Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption.

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).

Building and Breaking the Cell Wall in Four Acts: A Kinesthetic and Tactile Role-Playing Exercise for Teaching Beta-Lactam Antibiotic Mechanism of Action and Resistance.

"Building and breaking the cell wall" is designed to review the bacterial cell envelope, previously learned in lower-division biology classes, while introducing new topics such as antibiotics and bacterial antibiotic resistance mechanisms. We developed a kinesthetic and tactile modeling activity where students act as cellular components and construct the cell wall. In the first two acts, students model a portion of the gram-positive bacterial cell envelope and then demonstrate in detail how the peptidoglycan is formed. Act III involves student demonstration of the addition of ß-lactam antibiotics to the environment and how they inhibit the formation of peptidoglycan, thereby preventing bacterial replication. Using Staphylococcus aureus as a model for gram-positive bacteria, students finish the activity (Act IV) by acting out how S. aureus often becomes resistant to ß-lactam antibiotics. A high level of student engagement was observed, and the activity received positive feedback. In an assessment administered prior to and two months after the activity, significant improvements in scores were observed (p < 0.0001), demonstrating increased understanding and retention. This activity allows students to (i) visualize, role play, and kinesthetically "build" the cell envelope and form the peptidoglycan layer, (ii) understand the mechanism of action for ß-lactam antibiotics, as well as how gene acquisition and protein changes result in resistance, and (iii) work cooperatively and actively to promote long-term retention of the subject material.

Cytoplasmic transport and nuclear import of plasmid DNA.

Productive transfection and gene transfer require not simply the entry of DNA into cells and subsequent transcription from an appropriate promoter, but also a number of intracellular events that allow the DNA to move from the extracellular surface of the cell into and through the cytoplasm, and ultimately across the nuclear envelope and into the nucleus before any transcription can initiate. Immediately upon entry into the cytoplasm, naked DNA, either delivered through physical techniques or after disassembly of DNA-carrier complexes, associates with a large number of cellular proteins that mediate subsequent interactions with the microtubule network for movement toward the microtubule organizing center and the nuclear envelope. Plasmids then enter the nucleus either upon the mitotic disassembly of the nuclear envelope or through nuclear pore complexes in the absence of cell division, using a different set of proteins. This review will discuss our current understanding of these pathways used by naked DNA during the transfection process. While much has been elucidated on these processes, much remains to be discerned, but with the development of a number of model systems and approaches, great progress is being made.

Featured Article: Electroporation-mediated gene delivery of surfactant protein B (SP-B) restores expression and improves survival in mouse model of SP-B deficiency.

Surfactant Protein B Deficiency is a rare but lethal monogenetic, congenital lung disease of the neonate that is unresponsive to any treatment except lung transplantation. Based on the potential that gene therapy offers to treat such intractable diseases, our objective was to test whether an electroporation-based gene delivery approach could restore surfactant protein B expression and improve survival in a compound knockout mouse model of surfactant protein B deficiency. Surfactant protein B expression can be shut off in these mice upon withdrawl of doxycycline, resulting in decreased levels of surfactant protein B within four days and death due to lung dysfunction within four to seven days. Control or one of several different human surfactant protein B-expressing plasmids was delivered to the lung by aspiration and electroporation at the time of doxycycline removal or four days later. Plasmids expressing human surfactant protein B from either the UbC or CMV promoter expressed surfactant protein B in these transgenic mice at times when endogenous surfactant protein B expression was silenced. Mean survival was increased 2- to 5-fold following treatment with the UbC or CMV promoter-driven plasmids, respectively. Histology of all surfactant protein B treated groups exhibited fewer neutrophils and less alveolar wall thickening compared to the control groups, and electron microscopy revealed that gene transfer of surfactant protein B resulted in lamellar bodies that were similar in the presence of electron-dense, concentric material to those in surfactant protein B-expressing mice. Taken together, our results show that electroporation-mediated gene delivery of surfactant protein B-expressing plasmids improves survival, lung function, and lung histology in a mouse model of surfactant protein B deficiency and suggest that this may be a useful approach for the treatment of this otherwise deadly disease. Impact statement Surfactant protein B (SP-B) deficiency is a rare but lethal genetic disease of neonates that results in severe respiratory distress with no available treatments other than lung transplantation. The present study describes a novel treatment for this disease by transferring the SP-B gene to the lungs using electric fields in a mouse model. The procedure is safe and results in enough expression of exogenous SP-B to improve lung histology, lamellar body structure, and survival. If extended to humans, this approach could be used to bridge the time between diagnosis and lung transplantation and could greatly increase the likelihood of affected neonates surviving to transplantation and beyond.

Leptomycin B alters the subcellular distribution of CRM1 (Exportin 1).

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 h, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB.