The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFβ3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion.

BackgroundIn humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFβ3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFβ3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFβ3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFβ3 knockout mice to identify TGFβ3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFβ3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5).ResultsThe overall transcriptome analysis of TGFβ3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFβ3+/− and TGFβ3−/− were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFβ3−/− mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFβ3−/− mice. When the significantly altered CP genes were overlaid with TGFβ signaling, all of these genes followed the Smad-dependent pathway.ConclusionsOur study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFβ3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFβ3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.