Optimization of hCFTR lung expression in mice using DNA nanoparticles.

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.

DNA nanoparticles: detection of long-term transgene activity in brain using bioluminescence imaging.

In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year.

Preparation and analysis of PEGylated poly-L-lysine DNA nanoparticles for gene delivery.

PEGylated poly-L-lysine DNA nanoparticles are composed of plasmid DNA compacted with poly-L-lysine conjugated with polyethylene glycol (PEG). They are soluble and stable in saline and tissue fluids, transfect nondividing cells, display minimal toxicity, and are effective in vivo and in humans. Moreover, they are easy to prepare in a reliable and reproducible fashion.

Compacted DNA nanoparticle gene transfer of GDNF to the rat striatum enhances the survival of grafted fetal dopamine neurons.

Previously it was established that infusion of glial cell line-derived neurotrophic factor (GDNF) protein into grafts of embryonic dopamine cells has a neurotrophic effect on the grafted cells. In this study we used a nonviral technique to transfer the gene encoding for GDNF to striatal cells. Plasmid DNA encoding for GDNF was compacted into DNA nanoparticles (DNPs) by 10 kDa polyethylene glycol (PEG)-substituted lysine 30-mers (CK(30)PEG10k) and then injected into the denervated striatum of rats with unilateral 6-hydroxydopamine lesions.

Long-term transgene expression in the central nervous system using DNA nanoparticles.

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids.

Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution.

A double-blind, dose escalation gene transfer trial was conducted in subjects with cystic fibrosis (CF), among whom placebo (saline) or compacted DNA was superfused onto the inferior turbinate of the right or left nostril. The vector consisted of single molecules of plasmid DNA carrying the cystic fibrosis transmembrane regulator- encoding gene compacted into DNA nanoparticles, using polyethylene glycol-substituted 30-mer lysine peptides. Entry criteria included age greater than 18 years, FEV1 exceeding 50% predicted, and basal nasal potential difference (NPD) isoproterenol responses (> or = -5 mV) that are typical for subjects with classic CF.

Minimal toxicity of stabilized compacted DNA nanoparticles in the murine lung.

Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 microg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice.

Nanoparticles of compacted DNA transfect postmitotic cells.

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase.

Sustained release of plasmid DNA using lipid microtubules and agarose hydrogel.

Non-viral gene therapy typically results in low transfection efficiencies and transient gene expression. To address these limitations, two sustained delivery systems capable of releasing functional, compacted DNA for over 50 days were designed. A luciferase plasmid was compacted with a polylysine-polyethylene glycol conjugate and released from agarose hydrogel and lipid microtubule-hydrogel delivery systems for over 50 days.