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. 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.

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. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.

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. These properties represent a substantial advance for nonviral gene transfer. This article describes the conjugation of methoxy-PEG-maleimide with the peptide CK(30) and the compaction of DNA with the resultant PEGylated polylysine. It also describes the analyses used to check the morphology and colloidal stability of the nanoparticles. These assays should be performed each time the nanoparticles are prepared because, although the compaction procedure is very reproducible, variations in product quality do sometimes occur (e.g., the particles are unstable or have an unacceptable morphology). Variations seem to happen most often when the source of plasmid or method of plasmid production is changed.

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. Sham controls were injected with saline. One week later, experimental animals received either a ventral mesencephalic (VM) tissue chunk graft or a cell suspension VM graft implanted into the denervated striatum. Grafts were allowed to integrate for 4-6 weeks and during this period we monitored spontaneous and drug-induced motor activity. Using stereological cell counting we observed a 16-fold increase in the number of surviving TH(+) cells within tissue chunk grafts placed into the striatum pretreated with pGDNF DNPs (14,923 +/- 4,326) when compared to grafts placed into striatum pretreated with saline (955 +/- 343). Similarly, we observed a sevenfold increase in the number of TH(+) cells within cell suspension grafts placed into the striatum treated with pGDNF DNPs when compared to cell suspension grafts placed into the saline dosed striatum. Behaviorally, we observed significant improvement in rotational scores and in spontaneous forepaw usage of the affected forelimb in grafted animals receiving prior treatment with compacted pGDNF DNPs when compared to grafted animals receiving saline control pretreatment. Data analysis for protein, morphological, and behavioral measures suggests that compacted pGDNF DNPs injected into the striatum can result in transfected cells overexpressing GDNF protein at levels that provide neurotrophic support for grafted embryonic dopamine neurons.

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. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

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. Twelve subjects were enrolled: 2 in dose level I (DLI) (0.8 mg DNA), 4 in DLII (2.67 mg), and 6 in DLIII (8.0 mg). The primary trial end points were safety and tolerability, and secondary gene transfer end points were assessed. In addition to routine clinical assessments and laboratory tests, subjects were serially evaluated for serum IL-6, complement, and C-reactive protein; nasal washings were taken for cell counts, protein, IL-6, and IL-8; and pulmonary function and hearing tests were performed. No serious adverse events occurred, and no events were attributed to compacted DNA. There was no association of serum or nasal washing inflammatory mediators with administration of compacted DNA. Day 14 vector polymerase chain reaction analysis showed a mean value in DLIII nasal scraping samples of 0.58 copy per cell. Partial to complete NPD isoproterenol responses were observed in eight subjects: one of two in DLI, three of four in DLII, and four of six in DLIII. Corrections persisted for as long as 6 days (1 subject to day 28) after gene transfer. In conclusion, compacted DNA nanoparticles can be safely administered to the nares of CF subjects, with evidence of vector gene transfer and partial NPD correction.

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 containing 10 microg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 microg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 microg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 microg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 microg and a very modest effect, which is not limiting, at a dose of 100 microg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration.

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. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.

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. The released DNA was characterized structurally using sedimentation, electron microscopy, and serum stability, and functionally using in vitro transfections. The released DNA retained its physical compaction and nuclease resistance and was converted from supercoiled to nicked and linear forms. Released compacted DNA produced significant gene expression in vitro, although at lower levels than freshly compacted DNA. Thus, hydrogels and lipid microtubules successfully provided the slow release of bioactive, compacted DNA.