Focused ultrasound enhances transgene expression of intranasal hGDNF DNA nanoparticles in the sonicated brain regions.

The therapeutic potential of many gene therapies is limited by their inability to cross the blood brain barrier (BBB). While intranasal administration of plasmid DNA nanoparticles (NPs) offers a non-invasive approach to bypass the BBB, it is not targeted to disease-relevant brain regions. Here, our goal was to determine whether focused ultrasound (FUS) can enrich intranasal delivery of our plasmid DNA NPs to target deeper brain regions, in this case the regions most affected in Parkinson's disease. Combining FUS with intranasal administration resulted in enhanced delivery of DNA NPs to the rodent brain, by recruitment and transfection of microglia. FUS increased transgene expression by over 3-fold after intranasal administration compared to intravenous administration. Additionally, FUS with intranasal delivery increased transgene expression in the sonicated hemisphere by over 80%, altered cellular transfection patterns at the sonication sites, and improved penetration of plasmid NPs into the brain parenchyma (with a 1-fold and 3-fold increase in proximity of transgene expression to neurons in the forebrain and midbrain respectively, and a 40% increase in proximity of transgene expression to dopaminergic neurons in the substantia nigra). These results provide evidence in support of using FUS to improve transgene expression after intranasal delivery of non-viral gene therapies.

Remote magnetic navigation compared to contemporary manual techniques for the catheter ablation of ventricular arrhythmias in structural heart disease.

BACKGROUND: There are limited data comparing remote magnetic navigation (RMN) to contemporary techniques of manual-guided ventricular arrhythmia (VA) catheter ablation. OBJECTIVES: We compared acute and long-term outcomes of VA ablation guided by either RMN or contemporary manual techniques in patients with structural heart disease. METHODS: From 2010-2019, 192 consecutive patients, with ischemic cardiomyopathy (ICM) or non-ischemic cardiomyopathy (NICM) underwent catheter ablation for sustained ventricular tachycardia (VT) or premature ventricular complexes (PVCs), using either RMN (n = 60) or manual (n = 132) guided techniques. Acute success and VA-free survival were compared. RESULTS: In ICM, acute procedural success was comparable between the 2 techniques (manual 43.5% vs. RMN 29%, P = 0.11), as was VA-free survival (manual 83% vs. RMN 74%, P = 0.88), and survival free from cardiac transplantation and all-cause mortality (manual 88% vs. RMN 87%, P = 0.47), both at 12-months after final ablation. In NICM, manual compared to RMN guided, had superior acute procedural success (manual 46% vs. RMN 19%, P = 0.003) and VA-free survival 12-months after final ablation (manual 79% vs. RMN 41%, P = 0.004), but comparable survival free from cardiac transplantation and all-cause mortality 12-months after final ablation (manual 95% vs. RMN 90%, P = 0.52). Procedural duration was shorter in both subgroups undergoing manual guided ablation, whereas fluoroscopy dose and complication rates were comparable. CONCLUSION: RMN provides similar outcomes to manual ablation in patients with ICM. In NICM however, acute success, and long-term VA-free survival was better with manual ablation. Prospective, multi-centre randomised trials comparing contemporary manual and RMN systems for VA catheter ablation are needed.

Low Incidence of Ischemic Stroke Associated With Thrombus Aspiration in STEMI Patients Undergoing Primary PCI.

BACKGROUND: Thrombus aspiration (TA) during primary percutaneous coronary intervention (PPCI) for ST-segment elevation myocardial infarction (STEMI) was recommended to minimize distal embolization and to reduce thrombus burden prior to PPCI. Subsequent randomized trials showed no mortality benefit from TA and suggested an increased risk of stroke up to 180 days following TA, although it was not obvious that the procedure alone caused the strokes. METHODS AND RESULTS: This study retrospectively analyzed the periprocedural stroke rate in a series of STEMI patients treated with TA and PPCI at a single, large, tertiary hospital, where a rigorous uniform protocol of aspiration was used in all patients. Of 3734 patients, 1404 patients (38%; group 1) underwent TA as part of the PPCI procedure and 2330 patients (62%; group 2) did not undergo TA. There were no significant clinical differences between the 2 groups. In total, there were 20 strokes (0.54%), with 3 (0.2%) occurring in group 1, and 17 (0.7%) occurring in group 2 (P=.04). The majority of strokes occurred within 5 days of the procedure, and 3 (0.08%) were hemorrhagic. There were 22 intraprocedural deaths (0.6%), related to cardiogenic shock. There were no intraprocedural strokes. CONCLUSIONS: Very low stroke rates immediately post STEMI were seen in patients undergoing TA and PPCI in this real-world study. TA can be performed safely in STEMI patients undergoing PPCI with a short-term stroke risk equivalent to risk without TA. Further studies may be needed to explain the increased incidence of late stroke noted after TA and elucidate causative mechanisms.

Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) gene therapy could offer a disease-modifying treatment for Parkinson's disease (PD). Here, we report that plasmid DNA nanoparticles (NPs) encoding human GDNF administered intranasally to rats induce transgene expression in the brain and protect dopamine neurons in a model of PD. To first test whether intranasal administration could transfect cells in the brain, rats were sacrificed 1 week after intranasal pGDNF NPs or the naked plasmid. GDNF ELISA revealed significant increases in GDNF expression throughout the brain for both treatments. To assess whether expression was sufficient to protect dopamine neurons, naked pGDNF and pGDNF DNA NPs were given intranasally 1 week before a unilateral 6-hydroxydopamine lesion in a rat model of PD. Three to four weeks after the lesion, amphetamine-induced rotational behavior was reduced, and dopaminergic fiber density and cell counts in the lesioned substantia nigra and nerve terminal density in the lesioned striatum were significantly preserved in rats given intranasal pGDNF. The NPs afforded a greater level of neuroprotection than the naked plasmid. These results provide proof-of-principle that intranasal administration of pGDNF DNA NPs can offer a non-invasive, non-viral gene therapy approach for early-stage PD.

Intranasal delivery of hGDNF plasmid DNA nanoparticles results in long-term and widespread transfection of perivascular cells in rat brain.

The intranasal route of administration allows large therapeutics to circumvent the blood-brain barrier and be delivered directly to the CNS. Here we examined the distribution and pattern of cellular transfection, and the time course of transgene expression, in the rat brain after intranasal delivery of plasmid DNA nanoparticles (NPs) encoding hGDNF fused with eGFP. Intranasal administration of these NPs resulted in transfection and transgene expression throughout the rat brain, as indicated by eGFP ELISA and eGFP-positive cell counts. Most of the transfected cells were abluminal and immediately adjacent to capillaries and are likely pericytes, consistent with their distribution by perivascular transport. Intranasal administration of these plasmid DNA NPs resulted in significant, long-term transgene expression in rat brain, with highest levels at 1 week and continued expression for 6 months. These results provide evidence in support of intranasal DNA NPs as a non-invasive, long-term gene therapy approach for various CNS disorders.

Optimizing Non-viral Gene Therapy Vectors for Delivery to Photoreceptors and Retinal Pigment Epithelial Cells.

Considerable progress has been made in the design and delivery of non-viral gene therapy vectors, but, like their viral counterparts, therapeutic levels of transgenes have not met the requirements for successful clinical applications so far. The biggest advantage of polymer-based nanoparticle vectors is the ease with which they can be modified to increase their ability to penetrate the cell membrane and target specific cells by simply changing the formulation of the nanoparticle compaction. We took advantage of this characteristic to improve transfection rates of our particles to meet the transgene levels which will be needed for future treatment of patients. For this study, we successfully investigated the possibility of our established pegylated polylysine particles to be administered via intravitreal rather than subretinal route to ease the damage during injection. We also demonstrated that our particles are flexible enough to sustain changes in the formulation to accommodate additional targeting sequences without losing their efficiency in transfecting neuronal cells in the retina. Together, these results give us the opportunity to even further improve our particles.

DNA nanoparticles are safe and nontoxic in non-human primate eyes.

INTRODUCTION: DNA nanoparticles (NPs) comprising polylysine conjugated to polyethylene glycol efficiently target murine photoreceptors and the retinal pigment epithelium (RPE) and lead to long-term phenotypic improvement in models of retinal degeneration. Advancing this technology requires testing in a large animal model, particularly with regard to safety. So, herein we evaluate NPs in non-human primates (baboon). METHODS AND RESULTS: NPs with plasmids carrying GFP and a ubiquitous, RPE-specific, or photoreceptor-specific promoter were delivered by either subretinal or intravitreal injection. We detected GFP message and protein in the retina/RPE from eyes dosed with NPs carrying ubiquitously expressed and RPE-specific vectors, and GFP message in eyes injected with NPs carrying photoreceptor-specific vectors. Importantly, we observed NP DNA in the retina/RPE following intravitreal injection, indicating the inner limiting membrane does not prevent NP diffusion into the outer retina. We did not observe any adverse events in any baboon, and there were no NP-associated changes in retinal function. Furthermore, no systemic or local inflammatory reaction to the vectors/injections was observed, and no NP DNA was found outside the eye. CONCLUSION: Taken together with the well-established rodent safety and efficacy data, these findings suggest that DNA NPs may be a safe and potentially clinically viable nonviral ocular therapy platform for retinal diseases.

Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy.

We recently reported that the Ins2(Akita) mouse is a good model for late-onset diabetic retinopathy. Here, we investigated the effect of miR200-b, a potential anti-angiogenic factor, on VEGF receptor 2 (VEGFR-2) expression and to determine the underlying angiogenic response in mouse endothelial cells, and in retinas from aged Ins2(Akita) mice. MiR200-b and its native flanking sequences were amplified and cloned into a pCAG-eGFP vector directed by the ubiquitous CAG promoter (namely pCAG-miR200-b-IRES-eGFP). The plasmid was compacted by CK30PEG10K into DNA nanoparticles (NPs) for in vivo delivery. Murine endothelial cell line, SVEC4-10, was first transfected with the plasmid. The mRNA levels of VEGF and VEGFR-2 were quantified by qRT-PCR and showed significant reduction in message expression compared with lipofectamine-transfected cells. Transfection of miR200-b suppressed the migration of SVEC4-10 cells. There was a significant inverse correlation between the level of expression of miR200-b and VEGFR-2. Intravitreal injection of miR200-b DNA NPs significantly reduced protein levels of VEGFR-2 as revealed by western blot and markedly suppressed angiogenesis as evaluated by fundus imaging in aged Ins2(Akita) mice even after 3months of post-injection. These findings suggest that NP-mediated miR200-b delivery has negatively regulated VEGFR-2 expression in vivo.

Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype.

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.

Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases.

Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy.

S/MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA.

Mutations in genes in the retinal pigment epithelium (RPE) cause or contribute to debilitating ocular diseases, including Leber's congenital amaurosis (LCA). Genetic therapies, particularly adeno-associated viruses (AAVs), are a popular choice for monogenic diseases; however, the limited payload capacity of AAVs combined with the large number of retinal disease genes exceeding that capacity make the development of alternative delivery methods critical. Here, we test the ability of compacted DNA nanoparticles (NPs) containing a plasmid with a scaffold matrix attachment region (S/MAR) and vitelliform macular dystrophy 2 (VMD2) promoter to target the RPE, drive long-term, tissue-specific gene expression and mediate proof-of-principle rescue in the rpe65(-/-) model of LCA. We show that the S/MAR-containing plasmid exhibited reporter gene expression levels several fold higher than plasmid or NPs without S/MARs. Importantly, this expression was highly persistent, lasting up to 2 years (last timepoint studied). We therefore selected this plasmid for testing in the rpe65(-/-) mouse model and observe that NP or plasmid VMD2-hRPE65-S/MAR led to structural and functional improvements in the LCA disease phenotype. These results indicate that the non-viral delivery of hRPE65 vectors can result in persistent, therapeutically efficacious gene expression in the RPE.

Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice.

Mutations in the photoreceptor-specific flippase ABCA4 are associated with Stargardt disease and many other forms of retinal degeneration that currently lack curative therapies. Gene replacement is a logical strategy for ABCA4-associated disease, particularly given the current success of traditional viral-mediated gene delivery, such as with adeno-associated viral (AAV) vectors. However, the large size of the ABCA4 cDNA (6.8 kbp) has hampered progress in the development of genetic treatments. Nonviral DNA nanoparticles (NPs) can accommodate large genes, unlike traditional viral vectors, which have capacity limitations. We utilized an optimized DNA NP technology to subretinally deliver ABCA4 to Abca4-deficient mice. We detected persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of functional and structural Stargardt phenotypes, such as improved recovery of dark adaptation and reduced lipofuscin granules. These data suggest that DNA NPs may be an excellent, clinically relevant gene delivery approach for genes too large for traditional viral vectors.

Direct gene transfer with compacted DNA nanoparticles in retinal pigment epithelial cells: expression, repeat delivery and lack of toxicity.

AIM: To evaluate the safety of compacted DNA nanoparticles (NPs) in retinal pigment epithelial (RPE) cells. MATERIALS & METHODS: Enhanced GFP expression cassettes controlled by the RPE-specific vitelloform macular dystrophy promoter were constructed with and without a bacterial backbone and compacted into NPs formulated with polyethylene glycol-substituted lysine 30-mers. Single or double subretinal injections were administered in adult BALB/c mice. Expression levels of enhanced GFP, proinflammatory cytokines and neutrophil/macrophage mediators, and retinal function by electroretinogram were evaluated at different time-points postinjection. RESULTS: Immunohistochemistry and real-time PCR demonstrated that NPs specifically transfect RPE cells at a higher efficiency than naked DNA and similar results were observed after the second injection. At 6 h postinjections, a transient inflammatory response was observed in all cohorts, including saline, indicating an adverse effect to the injection procedure. Subsequently, no inflammation was detected in all experimental groups. CONCLUSION: This study demonstrates the safety and efficacy of NP-mediated RPE gene transfer therapy following multiple subretinal administrations.

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.

Highly compacted DNA nanoparticles with low MW PEG coatings: in vitro, ex vivo and in vivo evaluation.

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10kDa polyethylene glycol (CK(30)PEG(10k)), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK(30)PEG(10k) DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5kDa), whereas nanoparticles with 10kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK(30)PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK(30)PEG DNA nanoparticles prepared using various PEG MWs. CK(30)PEG(10k) and CK(30)PEG(5k) formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.

Enhancement of airway gene transfer by DNA nanoparticles using a pH-responsive block copolymer of polyethylene glycol and poly-L-lysine.

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK(30)), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH(12)K(18). Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH(12)K(18) to levels comparable with branched polyethyleneimine. PEG-CH(12)K(18) compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK(30) DNA nanoparticles. PEG-CH(12)K(18) DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH(12)K(18) DNA nanoparticles improved the in vitro gene transfer by ~20-fold over PEG-CK(30) DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.

Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells.

Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ~3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases.

N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

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.