Electroporation-mediated Gene Transfer Research Articles

MED13L and its disease-associated variants influence the dendritic development of cerebral cortical neurons in the mammalian brain.

The mediator complex comprises multiple subcellular subunits that collectively function as a molecular interface between RNA polymerase II and gene-specific transcription factors. Recently, genetic variants to one subunit of the complex, known as MED13L (mediator complex subunit 13 like), have been implicated in syndromic intellectual disability and distinct facial features, frequently accompanied by congenital heart defects. We investigated the impact of five disease-associated MED13L variants on the subcellular localization and biochemical stability of MED13L protein in vitro and in vivo. In overexpression assays using cortical neurons from embryonic mouse cerebral cortices transduced by in utero electroporation-mediated gene transfer, we found that mouse orthologues of human MED13L-p.P866L and -p.T2162M missense variants accumulated in the nucleus, while the p.S2163L and p.S2177Y variants were diffusely distributed in the cytoplasm. In contrast, we found that the p.Q1922* truncation variant was barely detectable in transduced cells, a phenotype reminiscent of this variant that results in MED13L haploinsufficiency in humans. Next, we analyzed these variants for their effects on neuronal migration, dendritic growth, spine morphology, and axon elongation of cortical neurons in vivo. There, we found that overexpression of the p.P866L variant resulted in reduced number and length of dendrites of cortical layer II/III pyramidal neurons. Furthermore, we show that mMED13L-knockdown abrogated dendritic growth in vivo, and this effect was significantly rescued by co-electroporation of an RNAi-resistant mMED13L, but weakly by the p.T2162M variant, and not at all by the p.S2163L variant. However, overexpression of the p.S2163L variant inhibited mature dendritic spine formation in vivo. Expression of each of the 5 variants did not affect neuronal cell migration and callosal axon elongation in vivo. Taken together, our results demonstrate that MED13L expression is relevant to corticogenesis and influences the dendritic branching characteristics of cortical excitatory neurons. Our study also suggests that disease-associated MED13L variants may directly cause morphological and functional defects in cortical neurons in different ways.

Anti-Fibrotic Effect of SDF-1β Overexpression in Bleomycin-Injured Rat Lung

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1β on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1β or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1β gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1β-induced apoptosis of myofibroblasts; moreover, SDF-1β overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1β overexpression and suggests SDF-1β as a potential new approach for the treatment of lung fibrosis.

Impaired Function of PLEKHG2, a Rho-Guanine Nucleotide-Exchange Factor, Disrupts Corticogenesis in Neurodevelopmental Phenotypes.

Homozygosity of the p.Arg204Trp variation in the Pleckstrin homology and RhoGEF domain containing G2 (PLEKHG2) gene, which encodes a Rho family-specific guanine nucleotide-exchange factor, is responsible for microcephaly with intellectual disability. However, the role of PLEKHG2 during neurodevelopment remains unknown. In this study, we analyzed mouse Plekhg2 function during cortical development, both in vitro and in vivo. The p.Arg200Trp variant in mouse (Plekhg2-RW), which corresponds to the p.Arg204Trp variant in humans, showed decreased guanine nucleotide-exchange activity for Rac1, Rac3, and Cdc42. Acute knockdown of Plekhg2 using in utero electroporation-mediated gene transfer did not affect the migration of excitatory neurons during corticogenesis. On the other hand, silencing Plekhg2 expression delayed dendritic arbor formation at postnatal day 7 (P7), perhaps because of impaired Rac/Cdc42 and p21-activated kinase 1 signaling pathways. This phenotype was rescued by expressing an RNAi-resistant version of wildtype Plekhg2, but not of Plekhg2-RW. Axon pathfinding was also impaired in vitro and in vivo in Plekhg2-deficient cortical neurons. At P14, knockdown of Plekhg2 was observed to cause defects in dendritic spine morphology formation. Collectively, these results strongly suggest that PLEKHG2 has essential roles in the maturation of axon, dendrites, and spines. Moreover, impairment of PLEKHG2 function is most likely to cause defects in neuronal functions that lead to neurodevelopmental disorders.

Gene transfer of MRCK\u03b1 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 β1 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 β1 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 β1 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.

Gene Delivery of MRCKα Repairs the Alveolar‐Capillary Barrier and Rescues the Experimental Lipopolysaccharide‐Induced Acute Lung Injury

Rationale Acute Respiratory Distress Syndrome (ARDS) is a devastating condition which is characterized by the pulmonary accumulation of protein-rich edema fluids and insufficient gas exchange, leading to acute hypoxemic respiratory failure. During ARDS, the net fluid clearance is undermined not only due to the impaired alveolar fluid clearance (AFC) which osmotically drives water following the ion gradient out of alveoli, but also the disrupted alveolar-capillary barrier. The previous data from our lab indicated that electroporation-mediated gene delivery of the β1 subunit of the Na+, K+-ATPase into mouse lungs treats LPS induced acute lung injury (ALI), showing significantly decreased lung edema, and improved overall outcome of lung injury. Overexpression of β1 increased AFC in healthy and injured murine lungs, and more importantly, gene delivery of β1 restored pulmonary barrier function, demonstrated by upregulated tight junction (TJ) proteins, and decreased lung permeability, total protein and cellularity in BAL fluid. MRCKα (CDC42 binding protein kinase alpha) was identified by our lab as an interacting partner of the β1 subunit through mass spectrometry. MRCKα is a Rho GTPase effector kinase, regulating diverse cell behaviors, including junction formation. Our previous in vitro data indicate that MRCKα mediated β1’s upregulation of TJ proteins and epithelial barrier integrity. Overexpression of MRCKα alone sufficiently increased the trans-epithelial electrical resistance (TEER) in cell culture. Thus, it is intriguing to investigate whether MRCKα has the therapeutic potential to treat LPS induced ALI by restoring pulmonary barrier function in vivo. This study tested whether electroporation-mediated gene transfer of MRCKα could upregulate TJ protein and barrier function and treat LPS induced ALI in mice. Methods ALI was induced by LPS intratracheal administration and 24 hours later, plasmids encoding MRCKα, β1- Na+, K+-ATPase, or nothing (control) were delivered individually or in combination to mouse lungs by oropharyngeal aspiration and electroporation. Two days after gene delivery, various endpoint assays were performed to evaluate lung edema, permeability, inflammation and histological injury. In addition, AFC was performed on mice with MRCKα overexpression to evaluate the fluid clearance. Results Gene transfer of MRCKα alone did not enhance AFC, but alone or in combination with β1- Na+, K+-ATPase attenuated LPS-increased Wet/Dry ratio, lung leakage, and BAL cellularity and protein concentration, restored TJ protein expression, and improved overall outcome of lung injury. Conclusions Gene delivery ofMRCKα repair alveolar capillary barrier integrity without promoting ion transport, and could benefit the pre-injured lungs by reducing pulmonary edema, restoring lung barrier function and reducing inflammation. Moreover, the data also suggest that improving barrier function alone may be of equal or even more benefit than improving alveolar fluid clearance in order to treat ALI/ARDS.

Efficacy of Combined in-vivo Electroporation-Mediated Gene Transfer of VEGF, HGF, and IL-10 on Skin Flap Survival, Monitored by Label-Free Optical Imaging: A Feasibility Study.

Preventing surgical flaps necrosis remains challenging. Laser Doppler imaging and ultrasound can monitor blood flow in flap regions, but they do not directly measure the cellular response to ischemia. The study aimed to investigate the efficacy of synergistic in-vivo electroporation-mediated gene transfer of interleukin 10 (IL-10) with either hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) on the survival of a modified McFarlane flap, and to evaluate the effect of the treatment on cell metabolism, using label-free fluorescence lifetime imaging. Fifteen male Wistar rats (290–320 g) were randomly divided in three groups: group-A (control group) underwent surgery and received no gene transfer. Group-B received electroporation mediated hIL-10 gene delivery 24 h before and VEGF gene delivery 24 h after surgery. Group-C received electroporation mediated hIL-10 gene delivery 24 h before and hHGF gene delivery 24 h after surgery. The animals were assessed clinically and histologically. In addition, label-free fluorescence lifetime imaging was performed on the flap. Synergistic electroporation mediated gene delivery significantly decreased flap necrosis (P = 0.0079) and increased mean vessel density (P = 0.0079) in treatment groups B and C compared to control group-A. NADH fluorescence lifetime analysis indicated an increase in oxidative phosphorylation in the epidermis of the group-B (P = 0.039) relative to controls. These findings suggested synergistic in-vivo electroporation-mediated gene transfer as a promising therapeutic approach to enhance viability and vascularity of skin flap. Furthermore, the study showed that combinational gene therapy promoted an increase in tissue perfusion and a relative increase in oxidative metabolism within the epithelium.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice.

Rationale: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. Methods: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. Results: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. Conclusion: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.

The Mouse Levator Auris Longus Muscle: An Amenable Model System to Study the Role of Postsynaptic Proteins to the Maintenance and Regeneration of the Neuromuscular Synapse.

The neuromuscular junction (NMJ) is the peripheral synapse that controls the coordinated movement of many organisms. The NMJ is also an archetypical model to study synaptic morphology and function. As the NMJ is the primary target of neuromuscular diseases and traumatic injuries, the establishment of suitable models to study the contribution of specific postsynaptic muscle-derived proteins on NMJ maintenance and regeneration is a permanent need. Considering the unique experimental advantages of the levator auris longus (LAL) muscle, here we present a method allowing for efficient electroporation-mediated gene transfer and subsequent detailed studies of the morphology and function of the NMJ and muscle fibers. Also, we have standardized efficient facial nerve injury protocols to analyze LAL muscle NMJ degeneration and regeneration. Our results show that the expression of a control fluorescent protein does not alter either the muscle structural organization, the apposition of the pre- and post-synaptic domains, or the functional neurotransmission parameters of the LAL muscle NMJs; in turn, the overexpression of MuSK, a major regulator of postsynaptic assembly, induces the formation of ectopic acetylcholine receptor clusters. Our NMJ denervation experiments showed complete reinnervation of LAL muscle NMJs four weeks after facial nerve injury. Together, these experimental strategies in the LAL muscle constitute effective methods to combine protein expression with accurate analyses at the levels of structure, function, and regeneration of the NMJ.

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts

BackgroundGene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments.ResultsHere, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 106 cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions.ConclusionsThe optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function

The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporation-mediated transuterine gene transfer of Connexin30, utilizing a monophasic pulse into Connexin30−/− mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. However, it is not clear whether supplementary gene transfer can rescue significant morphological changes, caused by genetic deficits. In addition, with the transuterine gene transfer technique utilized in our previous report, the survival rate of embryos and their mothers after treatment was low, which became a serious obstacle for effective in vivo experiments. Here, we set out to elucidate the feasibility of supplementation therapy in Slc26a4 deficient mice, utilizing biphasic pulses, optimized by modifying pulse conditions. Modification of the biphasic pulse conditions during electroporation increased the survival rate. In addition, supplementation of the target gene cDNA into the otocysts of homozygous Slc24a4 knockout mice significantly prevented enlargement of the endolymphatic space in the inner ear areas; moreover, it rescued hearing and vestibular function of mice in vivo.

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain.

In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain of Xenopus tadpoles by bulk electroporation-mediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications. Bilateral EM gene transfer allows for transfection of both sides of the brain, whereas unilateral EM gene transfer enables analysis of the effects of forced gene expression on one side of the brain, with the other side serving as the control.

Leptin Induces Mitosis and Activates the Canonical Wnt/β-Catenin Signaling Pathway in Neurogenic Regions of Xenopus Tadpole Brain.

In addition to its well-known role as an adipostat in adult mammals, leptin has diverse physiological and developmental actions in vertebrates. Leptin has been shown to promote development of hypothalamic circuits and to induce mitosis in different brain areas of mammals. We investigated the ontogeny of leptin mRNA, leptin actions on cell proliferation in the brain, and gene expression in the preoptic area/hypothalamus of tadpoles of Xenopus laevis. The level of leptin mRNA was low in premetamorphic tadpoles, but increased strongly at the beginning of metamorphosis and peaked at metamorphic climax. This increase in leptin mRNA at the onset of metamorphosis correlated with increased cell proliferation in the neurogenic zones of tadpole brain. We found that intracerebroventricular (i.c.v.) injection of recombinant Xenopus leptin (rxLeptin) in premetamorphic tadpoles strongly increased cell proliferation in neurogenic zones throughout the tadpole brain. We conducted gene expression profiling of genes induced at 2 h following i.c.v. injection of rxLeptin. This analysis identified 2,322 genes induced and 1,493 genes repressed by rxLeptin. The most enriched Kyoto Encyclopedia of Genes and Genomes term was the canonical Wnt/β-catenin pathway. Using electroporation-mediated gene transfer into tadpole brain of a reporter vector responsive to the canonical Wnt/β-catenin signaling pathway, we found that i.c.v. rxLeptin injection activated Wnt/β-catenin-dependent transcriptional activity. Our findings show that leptin acts on the premetamorphic tadpole brain to induce cell proliferation, possibly acting via the Wnt/β-catenin signaling pathway.

Developing guinea pig brain as a model for cortical folding.

The cerebral cortex in mammals, the neocortex specifically, is highly diverse among species with respect to its size and morphology, likely reflecting the immense adaptiveness of this lineage. In particular, the pattern and number of convoluted ridges and fissures, called gyri and sulci, respectively, on the surface of the cortex are variable among species and even individuals. However, little is known about the mechanism of cortical folding, although there have been several hypotheses proposed. Recent studies on embryonic neurogenesis revealed the differences in cortical progenitors as a critical factor of the process of gyrification. Here, we investigated the gyrification processes using developing guinea pig brains that form a simple but fundamental pattern of gyri. In addition, we established an electroporation-mediated gene transfer method for guinea pig embryos. We introduce the guinea pig brain as a useful model system to understand the mechanisms and basic principle of cortical folding.

Abstract 567: In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens

Abstract Cancer immunotherapy relies on presentation of shared- and neo- antigens from a patient's tumor cells for recognition and clearance by the immune system. However, the tumor microenvironment deploys multiple strategies to evade immune recognition and often remains non-immunogenic, which is one of the challenges that need to be addressed when designing new therapies. Among the strategies to increase a tumor's immunogenicity is their genetic manipulation in situ via expression of molecular chaperones, T cell costimulators and/or pro-inflammatory genes using DNA/RNA vectors packaged in oncolytic viruses, lipid based components or through electroporation. In vivo electroporation-mediated gene transfer of IL-12 triggers tumor regression and systemic anti-tumor immune responses in experimental mouse models and in patients, demonstrating the feasibility of this intratumoral (IT) gene-transfer technology. We set out to test whether intratumoral electroporation of Gp96-Ig/Fc-OX40L, a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression, would generate a robust anti-neoantigen CD8+ T cell response. Gp96-Ig/Fc-OX40L is a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression. To assess antigen-specific CD8+ expansion, mice were adoptively transferred with OT-I cells after B16.F10-ovalbumin cells were injected to generate primary and contralateral melanotic tumors. Contralateral tumors were monitored to assess whether a systemic CD8+ T cell response could be elicited following primary tumor electroporation. IT electroporation of DNA expressing Gp96-Ig/Fc-OX40L in the primary tumor triggered a significant expansion of antigen-specific OT-I cells, which was absent in control mice. Remarkably, increases in antigen-specific OT-I cells correlated with regression of both the treated primary and untreated contralateral tumors. We further validated our findings in a CT26 mouse colorectal cancer tumor model, in which the expression of Gp96-Ig/Fc-OX40L from electroporated DNA stimulated an expansion of antigen-specific CD8+ T cells and again led to regression of both the treated primary and untreated contralateral tumor. Our findings demonstrate that in situ manipulation of intratumoral cells to express Gp96-Ig/Fc-OX40L stimulates potent antigen-specific cross priming to tumor specific neoantigens that culminates in robust systemic anti-tumor response. These findings provide exciting proof-of-principal and warrant further investigation into the direct delivery of molecular chaperones such as Gp96-Ig/Fc-OX40L and/or pro-inflammatory molecules for elevating the immunogenicity of tumors for a potent anti-tumor CD8+ T cell response. Citation Format: Suresh de Silva, George Fromm, Jamil Haque, Jean S. Campbell, Robert H. Pierce, Taylor H. Schreiber. In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 567.

490. Electroporation-Mediated Gene Transfer of Caveolin-1 Protects from Bleomycin-Induced Pulmonary Fibrosis Through Regulating Activation of Inflammasome

Pulmonary fibrosis (PF) is a progressive chronic interstitial lung disease associated with high morbidity and mortality. It is characterized by increased deposition of extracellular matrix proteins and the accumulation of scar tissue in the lung interstitium, resulting from alveolar epithelial injury and the accumulation of inflammatory cells. Currently, there is no effective therapy. Gene therapy is a promising approach to treat a variety of lung diseases, including pulmonary fibrosis. Caveolin-1 (Cav-1) is a major component of caveolae, and has been found greatly reduced expression in the lungs of PF patients. Moreover, Cav-1-deficient mice develop spontaneous pulmonary fibrosis. To investigate if Cav-1 can protect from bleomycin-induced pulmonary fibrosis, we delivered plasmids expressing Cav-1 driven by the ubiquitin promoter using electroporation. PF was induced 2 days after gene transfer by intratracheal administration of bleomycin (2 unit/kg). We found that electroporation-mediated gene transfer of Cav-1 to the lung significantly protected from subsequent bleomycin-mediated fibrogenesis by histological analysis and the expression of alpha-smooth muscle actin (alpha-SMA) and collagen measurement. We also found attenuated recruitment of neutrophils and monocytes and/or macrophages in bronchoalveolar lavage (BAL) fluid after gene transfer of Cav-1 compared with vector control. The underlying mechanism was associated with reduced activation of the inflammasome by determining the expression of cleaved caspase-1 and IL-1 beta in both mouse models and cultured cells after overexpression of Cav-1. These results demonstrate that Cav-1 might be a potential target for treatment of pulmonary fibrosis.

A Mechanism to Enhance Cellular Responsivity to Hormone Action: Krüppel-Like Factor 9 Promotes Thyroid Hormone Receptor-β Autoinduction During Postembryonic Brain Development.

Thyroid hormone (TH) receptor (TR)-β (trb) is induced by TH (autoinduced) in Xenopus tadpoles during metamorphosis. We previously showed that Krüppel-like factor 9 (Klf9) is rapidly induced by TH in the tadpole brain, associates in chromatin with the trb upstream region in a developmental stage and TH-dependent manner, and forced expression of Klf9 in the Xenopus laevis cell line XTC-2 accelerates and enhances trb autoinduction. Here we investigated whether Klf9 can promote trb autoinduction in tadpole brain in vivo. Using electroporation-mediated gene transfer, we transfected plasmids into premetamorphic tadpole brain to express wild-type or mutant forms of Klf9. Forced expression of Klf9 increased baseline trb mRNA levels in thyroid-intact but not in goitrogen-treated tadpoles, supporting that Klf9 enhances liganded TR action. As in XTC-2 cells, forced expression of Klf9 enhanced trb autoinduction in tadpole brain in vivo and also increased TH-dependent induction of the TR target genes klf9 and thbzip. Consistent with our previous mutagenesis experiments conducted in XTC-2 cells, the actions of Klf9 in vivo required an intact N-terminal region but not a functional DNA binding domain. Forced expression of TRβ in tadpole brain by electroporation-mediated gene transfer increased baseline and TH-induced TR target gene transcription, supporting a role for trb autoinduction during metamorphosis. Our findings support that Klf9 acts as an accessory transcription factor for TR at the trb locus during tadpole metamorphosis, enhancing trb autoinduction and transcription of other TR target genes, which increases cellular responsivity to further TH action on developmental gene regulation programs.

A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.

359. Gene Transfer of the Na, K-ATPase beta1 Subunit and Occludin Using Electroporation Rescues Lipopolysaccaride (LPS)-Induced Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute respiratory failure and associated with diverse disorders. Gene therapy is a potentially powerful approach to treat ALI and ARDS through repair of alveolar epithelial function. We previously showed that electroporation-mediated gene transfer of the b1 subunit of the Na,K-ATPase to the lung not only increased alveolar fluid clearance, but also improved epithelium/endothelium barrier functions via up-regulation of tight junction proteins to provide partial treatment of lipopolysaccaride- (LPS-) injured lungs. To further investigate if we could improve treatment, we co-delivered plasmids expressing occludin, which is the one of major constituents of tight junctions located at the plasma membrane. Lung injury was induced 1 day before gene delivery by intratracheal administration of LPS (5 mg/kg) and assessed by wet-to-dry ratios, histological analysis, myeloperoxidase (MPO) measurement, and bronchoalveolar lavage (BAL) protein levels and cellularity. Delivery of b1-Na,K-ATPase and occludin alone or in combination using electroporation treated previously injured lungs in a mouse model, compared to the control plasmid pCDNA3. However, combined gene transfer of b1-Na,K-ATPase and occludin only provided similar treatment benefit to either gene delivery of b1-Na,K-ATPase or occludin alone although the expression of tight junction proteins had been increased. These results demonstrate that both b1-Na,K-ATPase and occludin regulate formation of tight junctions in severely injured lungs and thus may play a critical role in the treatment of ALI/ARDS. It indicates that epithelial or endothelial barrier functions may be involved in the treatment of ALI/ARDS.

Establishment of an in vivo electroporation method into postnatal newborn neurons in the dentate gyrus.

Electroporation-mediated gene transfer has been developed for the analysis of mammalian brain development in vivo. Indeed, in utero electroporation method is widely used for the investigation of the mouse embryonic cortical development while in vivo electroporation using neonatal mouse brain is employed for the analysis of the rostral migratory stream (RMS) and postnatal olfactory neurogenesis. In the present study, we established a stable gene-transfer method to dentate gyrus (DG) neurons by carefully determining the in vivo electroporation conditions, such as position and direction of electrode, voltage for electric pulses, and interval between electroporation and sample preparation. Consequently, GFP-positive cells in DG were observed to extend branched dendrites and long axons into the molecular layer and the hilus, respectively, 21 days after electrporation. They were morphologically identified as dentate granule neurons with many protrusions on dendrites, and some of them had wide head and thin neck that resembled matured mushroom spines. Expression of GFP in dentate neurons sustained for at least 9 months after electroporation under our experimental conditions. Taken together, the method developed here could be a powerful new tool for the analysis of the postnatal DG development.