Optimization of hCFTR Lung Expression in Mice Using DNA Nanoparticles

Abstract

Efficient and prolonged human cystic fibrosis transmembrane conductance regulator (hCFTR) expression is a major goal for cystic fibrosis (CF) lung therapy. A hCFTR expression plasmid was optimized as a payload for compacted DNA nanoparticles formulated with polyethylene glycol (PEG)-substituted 30-mer lysine peptides. A codon-optimized and CpG-reduced hCFTR synthetic gene (CO-CFTR) was placed in a polyubiquitin C expression plasmid. Compared to hCFTR complementary DNA (cDNA), CO-CFTR produced a ninefold increased level of hCFTR protein in transfected HEK293 cells and, when compacted as DNA nanoparticles, produced a similar improvement in lung mRNA expression in Balb/c and fatty acid binding protein promoter (FABP) CF mice, although expression duration was transient. Various vector modifications were tested to extend duration of CO-CFTR expression. A novel prolonged expression (PE) element derived from the bovine growth hormone (BGH) gene 3' flanking sequence produced prolonged expression of CO-CFTR mRNA at biologically relevant levels. A time course study in the mouse lung revealed that CO-CFTR mRNA did not change significantly, with CO-CFTR/mCFTR geometric mean ratios of 94% on day 2, 71% on day 14, 53% on day 30, and 14% on day 59. Prolonged CO-CFTR expression is dependent on the orientation of the PE element and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements.

Comparative Analysis of DNA Nanoparticles and AAVs for Ocular Gene Delivery

“…Although both the NPs and the AAV were generated from matched ITR plasmids, the NPs contain the entire plasmid while AAV production results in virions carrying only the expression cassette. Prokaryotic plasmid backbone element can influence gene expression [19], [36], and may promote NP-CBA-GFP silencing. A second contributing factor may be differential methylation or epigenetic state between AAV and NP DNA arising from the biological source of the DNA: bacteria for NPs and HEK cells for AAV.…”

“…These small NPs (minor diameter of 8–11 nm) are efficiently taken up into dividing and non-dividing cells and remain episomal [17], [18]. They were safe and effective in a human clinical trial for cystic fibrosis and are currently being employed in the lung, brain, and eye [15], [16], [19], [20], [21], [22], [23]. We have demonstrated that NPs can be safely used to target the photoreceptors and RPE cells without significant toxicity [21], [24], [25] and mediate improvement in the retinitis pigmentosa phenotype of the retinal degeneration slow ( rds +/− ) mouse model [15], [16].…”

Age and lesion-induced increases of GDNF transgene expression in brain following intracerebral injections of DNA nanoparticles

“…Detailed methodology for the pUL and pGDNF_1b plasmid construction is reported in previous publications (Fletcher et al, 2011, Padegimas et al, 2012). The transgene expression in pUL and pGDNF_1b plasmids are driven by the human polyubiquitin C ( UbC ) promoter.…”

Gene Therapy for Retinal Disease

“…Whereas these approaches utilise codon-modified gene replacements, complete codon optimisation of the transgene would not only be helpful in resistance to silencing but it may also enhance expression levels and therefore increase the chance and degree of physiological outcome (141). In vitro studies in HEK293 cells have shown that codon optimised genes can produce a nine-fold increase in protein level compared to the WT coding sequence (142). Whilst no retinal codon optimised comparisons have currently been reported, a codon optimised microdystrophin transgene showed significantly improved expression and physiological outcome in mdx mice following rAAV2/8 delivery (143).…”