Identification of an alveolar type I epithelial cell-specific DNA nuclear import sequence for gene delivery

Abstract

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.

Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy

“…Following successful endosomal escape and vector unpacking another significant barrier to gene delivery arises: nucleases present in the cytoplasm can degrade released NAs. For instance, several studies have demonstrated that the half-life of pDNA in the cytoplasm is in the range of 50-90 min [2,5,177]. Surprisingly, research on NA degradation in the cytosol remains rather limited to date, presumably because only few techniques are available to investigate this experimentally.…”

“…Gene therapy is defined as the delivery of therapeutic genes to target cells in order to replace or counteract a malfunctioning gene and has emerged as a promising strategy to cure a wide variety of inherited or acquired diseases at their genetic roots [1][2][3][4]. Besides the delivery of pDNA, several other nucleic acids (NAs), such as mRNA, siRNA and miRNA, have emerged as promising tools to modulate protein expression levels [5].…”

“…Here, the EC barriers are epithelial and extra factors such as the presence of mucus and surfactant play an important role. Together, these EC obstacles can lead to cargo degradation, rapid clearance of NPls or NPl immobilization, thereby rendering them ineffective [1,2]. Once past the EC barriers, several IC barriers obstruct the way to efficient gene delivery.…”

“…Once past the EC barriers, several IC barriers obstruct the way to efficient gene delivery. First, in order to reach the IC environment, NPls need to cross the plasma membrane [2,12,13]. Although physical methods, such as photoporation, electroporation or sonoporation, are sometimes used to provide direct access to the cell cytoplasm [7,14,15], non-viral NPls usually gain entrance to the cell by endocytosis [5,16].…”

“…Furthermore, the kinetics of cargo release from the carrier is an important consideration, since vector unpacking has been reported as another bottleneck for efficient transfection. Finally, while NAs such as siRNA, mRNA and miRNA have their site of action in the cytosol, pDNA needs to be delivered to the nucleus in order to be effective, thereby making the nuclear envelope a major barrier as well [2,12,13,18].…”