The SP-C promoter facilitates alveolar type II epithelial cell-specific plasmid nuclear import and gene expression.

Although nonviral gene therapy has great potential for use in the lung, the relative lack of cell-specific targeting has limited its applications. We have developed a new approach for cell-specific targeting based on selective nuclear import of plasmids in nondividing cells. Using a microinjection and in situ hybridization approach, we tested several potential DNA sequences for the ability to mediate plasmid nuclear import in alveolar type II epithelial (ATII) cells. Of these, only a sequence within the human surfactant protein C (SP-C) promoter was able to mediate nuclear localization of plasmid DNA specifically in ATII cells but not in other cell types. We have mapped the minimal import sequence to the proximal 318 nucleotides of the promoter, and demonstrate that binding sites for nuclear factor I, thyroid transcriptional factor 1, and GATA-binding protein 6 and the proteins themselves are required for import activity. Using intratracheal delivery of DNA followed by electroporation, we demonstrate that the SP-C promoter sequence will enhance gene expression specifically in ATII cells in mouse lung. This represents a new activity for the SP-C promoter, and thus ATII cell-specific nuclear import of DNA may prove to be a safe and effective method for targeted and enhanced gene expression in ATII cells.

Electroporation- and mechanical ventilation-mediated gene transfer to the lung.

Our laboratory has previously demonstrated that cytoplasmic trafficking and subsequent nuclear entry of nonviral plasmid DNA can be significantly enhanced through the application of cyclic stretch after transfection in vitro. In this study, we show that cyclic stretching of the murine lung using ventilation immediately after endotracheal administration and transthoracic electroporation of plasmid DNA increases exogenous gene expression up to fourfold in mice that were not ventilated after plasmid administration and transfection by electroporation in vivo. This increase is both time and sequence specific (that is, the ventilation must occur immediately after the transfection event). The ventilation-enhanced gene transfer is also amplitude dependent, confirming similar studies completed in vitro, and is mediated, at least in part, through the cytoplasmic tubulin deacetylase, HDAC6. Using immunohistochemistry, we show that this increase in expression is due to an increase in the number of cells expressing the exogenous protein rather than an increase in the amount of protein produced per cell. These studies show the potential mechanical stimulation has in vivo in significantly increasing nonviral DNA gene expression, and may ultimately pave the way for more successful clinical trials using this type of therapy in the future.

Emergence of functional neuromuscular junctions in an engineered, multicellular spinal cord-muscle bioactuator.

Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like "feet." We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a "spinobot" by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.

Ectopic expression of c-ski disrupts gastrulation and neural patterning in zebrafish.

The c-ski proto-oncogene encodes a transcriptional regulator that has been implicated in the development of different tissues at different times during vertebrate development. We identified two novel paralogues of the c-ski gene family, skiA and skiB in zebrafish (Danio rerio). The skiA protein is maternal and ubiquitous while skiB is zygotic. Overexpression of SkiA or SkiB disrupted gastrulation and resulted in a dorsalized phenotype. In situ analyses suggested that overexpression of Ski leads to a slight expansion of dorsal-axial mesoderm, diminishment or loss of ventral mesoderm and radialization of dorsal neuroectoderm. The dorsalized phenotype could be rescued by the ventral specifying factor, BMP4. These results provide evidence that Ski proteins participate in dorsal-ventral specification of both neuroectoderm and mesoderm.