Lipoplex-mediated gene delivery to the lung occurs within 60 minutes of intravenous administration.

We have defined the critical time period for gene delivery in the lung after intravenous administration of cationic lipoplex. We accomplished this through the displacement of intravenously injected cationic lipoplexes from the lungs by the subsequent administration of anionic liposomes. When reporter gene-bearing lipoplexes were injected intravenously and followed by anionic liposomes 5 min later, reporter gene expression was reduced up to 400-fold compared with animals into which lipoplex alone was administered. Administration of anionic liposomes 60-90 min after lipoplex injection yielded no significant reduction in lung transfection. When lipoplexes were disrupted 5 min after administration, the pulmonary distribution of the cationic lipid and DNA components was reduced by 80%. Lipids subsequently accumulated primarily in the liver, while the plasmid DNA constituent distributed into the blood and liver. As the interval between lipoplex and anionic liposome injection increased, the degree of lipoplex displacement from the lung decreased to such a point that, 60 min after lipoplex injection, the anionic liposome injection did not displace significant quantities of the lipoplex. We conclude that cationic lipid-DNA complexes can be disrupted in vivo via the administration of anionic liposomes; moreover, we have employed this phenomenon to demonstrate that transfectionally active DNA is taken up within 60 min of systemic lipoplex administration.     

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.     

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components.

We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the resulting fluorescent nuclei was used to define the relationship between cytoplasmic release of nucleic acids and gene expression. Endothelial cells were stained after i.v. administration, and epithelial cells were stained after i.t. administration. The delivery of nucleic acids was also more homogeneous with i.v. administration of lipoplex than with i.t. administration. After i.t. administration, it was notable that high concentrations of fluorescent nuclei correlated with low GFP expression. This suggested that toxicity was associated with high local concentrations of cationic lipoplexes. The ratio of GFP-expressing cells to fluorescent nuclei indicated that capillary endothelial cells were more efficient in gene expression per delivery event than were pulmonary epithelial cells. Thus, the greater gene expression efficiency of i.v. administered lipoplexes was due not only to the initial distribution but also to the greater efficiency of the vascular endothelial cells to appropriately traffic and express the foreign gene.     

Enhanced hepatocyte-selective in vivo gene expression by stabilized galactosylated liposome/plasmid DNA complex using sodium chloride for complex formation.

In this study, we demonstrated that the presence of an essential amount of sodium chloride (NaCl) during the formation of cationic liposome/plasmid DNA complexes (lipoplexes) stabilizes the lipoplexes according to the surface charge regulation (SCR) theory. Fluorescence resonance energy transfer analysis revealed that cationic liposomes in an SCR lipoplex (5 and 10 mM NaCl solution in lipoplex) increased fusion. Also, aggregation of SCR lipoplexes was significantly delayed after exposure to saline (150 mM NaCl) as a model of physiological conditions. After intraportal administration, the hepatic transfection activity of galactosylated SCR lipoplexes (5 and 10 mM NaCl solution in lipoplex) was approximately 10- to 20-fold higher than that of galactosylated conventional lipoplexes in mice. The transfection activity in hepatocytes of galactosylated SCR lipoplexes was significantly higher than that of conventional lipoplexes, and preexposure to competitive asialoglycoprotein-receptor blocker significantly reduced the hepatic gene expression, suggesting that hepatocytes are responsible for high hepatic transgene expression of the galactosylated SCR lipoplexes. Pharmacokinetic studies both in situ and in vivo demonstrated a higher tissue binding affinity and a greater expanse of intrahepatic distribution by galactosylated SCR lipoplexes. Moreover, enhanced transfection activity of galactosylated SCR lipoplexes was observed in HepG2 cells, and investigation of confocal microscopic images showed that the release of plasmid DNA in the cell was markedly accelerated. These characteristics partly explain the mechanism of enhanced in vivo transfection efficacy by galactosylated SCR lipoplexes. Hence, information in this study will be valuable for the future use, design, and development of ligand-modified lipoplexes for in vivo applications.     

Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic “cargo.” Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.     

Poly(cationic lipid)-mediated in vivo gene delivery to mouse liver

We have previously demonstrated that liposomes generated from poly(cationic lipid) (PCL) and cholesterol (Chol) have low cytotoxicity, are serum resistant, and display a transfection efficiency in vitro similar to commercially available cationic liposomes. Our in vivo experiments demonstrated that PCL-Chol liposomes bound much less avidly to serum proteins than did liposomes composed of 1,2-bis(dioleoyloxy)-3-(trimethylamonio)propane (DOTAP)-Chol or DOTAP-L-alpha dioleoyl phosphatidylethanolamine (DOPE). Injection of the lipoplexes (PCL-Chol+DNA) through the portal vein after partial hepatectomy (PH) led to much higher reporter gene expression (luciferase) in the liver than did naked DNA injection. Marked green fluorescent protein expression was visualized in almost all hepatocytes in the liver of mice receiving lipoplex injection, even in the absence of PH. Subcutaneous injection of thyroid hormone triiodothyromine (T(3)) significantly promoted hepatocyte regeneration and markedly enhanced PCL-Chol-mediated gene transfer in mouse liver when the lipoplex was administrated through either portal or tail vein. With T(3) pretreatment, PCL-Chol exerted a better gene transfer efficacy in mouse liver than DOTAP-Chol or DOTAP-DOPE. Two injections of lipoplexes through an indwelling catheter in the portal vein extended the transgene expression at a high level when T(3) injection was repeated. In conclusion, our findings demonstrate that the polymerized cationic liposomes are very stable in the blood and are effective agents for in vivo gene delivery, and that thyroid hormone administration offers a non-invasive approach to enhance liposome-mediated liver gene delivery.     

Efficient gene transfer into macrophages and dendritic cells by in vivo gene delivery with mannosylated lipoplex via the intraperitoneal route

In this study, we developed an antigen-presenting cell (APC)-selective intraperitoneal (i.p.) gene delivery system with mannosylated cationic liposomes (Man-liposomes)/plasmid DNA complex (Man-lipoplex). An in vitro study using cultured peritoneal macrophages demonstrated that Man-liposomes could transfect luciferase-encoding plasmid DNA (pCMV-Luc) more efficiently than cationic liposomes via a mannose receptor-mediated mechanism. In vivo gene transfection studies revealed that Man-lipoplex showed a higher gene expression in the liver, spleen, peritoneal exuded cells, and mesenteric lymph nodes than cationic liposomes/plasmid DNA complex (lipoplex) or naked pCMV-Luc after i.p. administration, and this gene expression lasted for at least 24 h. The transfection activity of Man-lipoplex after i.p. administration was significantly higher than that after i.v. gene delivery with the Man-liposomes we developed previously, indicating that gene delivery via the i.p. route seems to be an efficient approach for in vivo gene delivery to APCs. Furthermore, it was demonstrated that Man-lipoplex could enhance gene expression in both F4/80+ and CD11c+ cells in the spleen. These results show that gene delivery with Man-liposomes via the i.p. route could be an effective approach for APC-selective gene transfection.     

Intravascular and endobronchial DNA delivery to murine lung tissue using a novel, nonviral vector

Gene transfer to the lung can be achieved via either the airway or the pulmonary vasculature. We evaluated gene transfer and expression by intravascular and endobronchial routes, using DNA complexed with G9 PAMAM dendrimer or naked plasmid DNA. Intravascular tail vein delivery of dendrimer-complexed pCF1CAT plasmid resulted in high levels of transgene expression in the lung at 12 and 24 hr, followed by a second peak of expression 3 to 5 days after administration. After intravenous administration of the complexes, CAT expression was never observed in organs other than the lung. There were only minimal levels of CAT protein expressed in the lung after intravenous administration of naked plasmid DNA. Repeated intravascular doses of the dendrimer-complexed plasmid, administered four times at 4-day intervals, maintained expression at 15-25% of peak concentrations achieved after the initial dose. Endobronchial delivery of naked pCF1CAT plasmid produced significant amounts of CAT protein in the lung. Comparison of intratracheal and intranasal routes resulted in similar expression levels of CAT in the lung and trachea. However, in juxtaposition to vascular delivery, intranasal delivery of dendrimer-complexed plasmid DNA gave lower levels of CAT expression than that observed with naked plasmid DNA. In situ localization of CAT enzymatic activity suggested that vascular administration seemed to achieve expression in the lung parenchyma, mainly within the alveoli, while endobronchial administration primarily targeted bronchial epithelium. Our results show that intravenously administered G9 dendrimer is an effective vector for pulmonary gene transfer and that transgene expression can be prolonged by repeated administration of dendrimer-complexed DNA.     

Convective flow increases lipoplex delivery rate to in vitro cellular monolayers

The mass transport characteristics of cationic, nonviral liposome-DNA plasmid complexes (lipoplexes) were evaluated over a range of fluid shear stresses. The typical case of stagnant flow transfection was expanded to include controlled fluid convection provided by constant flow through a parallel plate flow chamber. Equations describing the transport of lipoplex by sedimentation and convection were derived from theory and solved numerically. Instantaneous lipoplex delivery rate and total lipoplex surface delivery during a 72-h transfection were estimated for two shear stress levels and for static conditions. Theory predicted that lipoplex is delivered to the cell surface more than 12- to 19-fold faster through the addition of convection, at least for physiologic shear stresses of 2.3-9.7 dyn/cm2, respectively. These calculations were tested experimentally using a cell line (ECV-304) transfected with fluorescently labeled plasmid DNA formulated into a lipoplex. Transfections were conducted during cellular exposure to the same known, uniform levels of fluid shear stress presumed in theoretical calculations. Lipoplex delivery was increased by more than nine-fold at 2.3 dyn/cm2 compared to the static case as assessed by flow cytometric measurement. Lipoplex delivery was modestly reduced at the highest fluid shear stress, to six-fold of the static case, consistent with the disruption of lipoplex-cell binding mediated by hydrodynamic forces. The complicated relationship between fluid convection and lipoplex delivery has important implications for nonviral gene therapy.     

Multiple systemic expression of human interferon-beta in mice can be achieved upon repeated administration of optimized pcTG90-lipoplex

The possibility of achieving multiple systemic expression of human interferon-beta in mice upon repeated intravenous administration of cationic liposome-DNA complex (lipoplex) was investigated. Lipoplexes containing the pentammonio lipid pcTG90 were first optimized by selecting the most efficient ratio of pcTG90 to phosphatidylethanolamine (DOPE) and the N/P ratio of cationic lipid nitrogen to DNA phosphate. Highest levels and reproducibility of gene expression were obtained using pcTG90/DOPE (1:2) liposomes complexed with the IFNB1 gene containing plasmid pTG14169 at a N/P ratio of 10. Following lipoplex administration, an early but transient human interferon-beta expression in serum was observed. Importantly, repeated systemic gene expression could be achieved upon re-administration with a minimal time interval of 14 days between two injections. For an interval period of 6 days, subsequent gene expression was inhibited by a first administration of lipoplexes containing either a luciferase reporter gene plasmid or an empty plasmid, but was not inhibited when free (non-complexed) plasmid pTG14169 was first injected. Multiple injections of pcTG90-lipoplex performed once every other month resulted in three subsequent peaks of systemic IFNB1 gene expression in mice. In conclusion, our study demonstrates the feasibility of expanding the therapeutic window of a cytokine using repetitive intravenous administration of lipoplex.     

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.     

Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid.

Simple, nontoxic, and pharmaceutically defined methods for genetic modification of respiratory tissues may enable development of a variety of molecular medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmonary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccination. "Free," "direct," or "naked" plasmid administration is a simple, apparently safe, and pharmaceutically defined gene delivery method. Murine, macaque, and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transduction, and specifically to provide a theoretical framework for developing a category of adjuvants, nuclease inhibitors, that augment the transfection activity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose escalation studies were performed using lung homogenate assays to characterize transfection. Potential toxicity was assessed histologically. The data indicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expression. The effective dose for ATA enhancement of direct lung transfection was 0.5 microg/g mouse weight, and the LD50 was approximately 6 microg/g. These findings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory transfection technology.     

Hereditary inclusion body myopathy: single patient response to intravenous dosing of GNE gene lipoplex

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult-onset myopathy due to mutations in the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Affected patients have no therapeutic options. We have previously demonstrated in preclinical testing the ability to safely correct GNE gene function through liposomal delivery of the wild-type GNE gene. Results were verified in a single patient treated by intravenous infusion of GNE gene lipoplex. A single patient (patient 001) with severe HIBM treated with a compassionate investigational new drug received seven doses of GNE gene lipoplex via intravenous infusion at the following doses: 0.4, 0.4, 1.0, 4.0, 5.0, 6.0, and 7.0?mg of DNA. GNE transgene expression, downstream induction of sialic acid, safety, and muscle function were evaluated. Transient low-grade fever, myalgia, tachycardia, transaminase elevation, hyponatremia, and hypotension were observed after infusion of each dose of GNE gene lipoplex. Quadriceps muscle expression of the delivered GNE, plasmid, and RNA was observed 24?hr after the 5.0-mg dose and at significantly greater levels 72?hr after the 7.0-mg infusion in comparison with expression in quadriceps muscle immediately before infusion. Sialic acid-related proteins were increased and stabilization in the decline of muscle strength was observed. We conclude that clinical safety and activity have been demonstrated with intravenous infusion of GNE gene lipoplex. Further assessment will involve a phase I trial of intravenous administration of GNE gene lipoplex in individuals with less advanced HIBM with more muscle function.     

Preclinical evaluation of inducible nitric oxide synthase lipoplex gene therapy for inhibition of stent-induced vascular neointimal lesion formation

Several reports have established the concept of nitric oxide synthase (NOS) gene transfer for inhibiting smooth muscle cell (SMC) proliferation after vascular injury. To minimize potential risks associated with viral gene transfer, we developed a liposome-based gene transfer approach employing inducible NOS (iNOS) overexpression for inhibition of stent-induced neointimal lesion formation. Therapeutic lipoplexes were transferred to femoral or coronary arteries of Goettingen minipigs, using the Infiltrator local drug delivery device. Efficiency of local iNOS lipoplex transfer was analyzed by iNOS-specific immunohistochemistry. NO-mediated inhibition of stent-induced neointimal lesion formation was analyzed by intravascular ultrasound (IVUS) and computerized morphometry. Gene transfer efficiency increased dose dependently to a maximum of 44.3 +/- 4.2% iNOS-positive vessel area (dose, 2 microg of iNOS lipoplex). Proliferating cell nuclear antigen (PCNA) expression of medial SMCs (immunohistochemistry) was inhibited significantly by transfer of 2 microg of iNOS lipoplexes (111 +/- 27 cells [iNOS] versus 481 +/- 67 cells [control; PCNA-positive medial cells]). IVUS analysis demonstrated that local transfer of iNOS lipoplexes resulted in a significant reduction of femoral in-stent plaque area (control, 40.85 +/- 6.37 mm(2); iNOS, 24.69 +/- 1.8 mm(2); p = 0.03). Coronary in-stent lesion formation was reduced by about 45% as determined by histologic morphometry (control, 4.0 +/- 0.29; iNOS, 2.2 +/- 0.30; p < 0.01). In conclusion, this study demonstrates that local intramural delivery of iNOS lipoplexes can exert therapeutic effects in inhibiting stent-induced neointimal lesion formation. Together with the nonviral character of this gene therapy approach, these findings may have important impact on the transition of NOS-based gene therapy to clinical practice.     

Enhanced gene expression in mouse muscle by sustained release of plasmid DNA using PPE-EA as a carrier

Delivery of plasmid DNA by nanoparticles improves the DNA bioavailability, for instance in intramuscular administration, by localizing the DNA in the muscle tissue. Extracellular sustained release of the DNA may lead to more prolonged transgene expression. The present study describes a novel controlled gene delivery system based on a water soluble and biodegradable polyphosphoester, poly(2-aminoethyl propylene phosphate) (PPE-EA). The polymer degraded in PBS at 37 degrees C through the cleavage of the backbone phosphate bonds, and it was synthesized with a relative high molecular weight to ensure a suitable hydrolytic stability as a gene carrier. The tissue response and cytotoxicity study demonstrated a better tissue compatibility of PPE-EA in mouse muscle compared with commonly used polyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently and protected DNA from nuclease and serum degradation. Sustained release of plasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EA degradation. The DNA release profiles appear to be predominantly controlled by carrier degradation and the release rate of plasmid could be adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P (amino to phosphate groups) ratio of 1, a 46% burst was observed for the first day, followed by about 4% release per day (24 microg DNA/day/mg of complex) for 12 days. Higher charge ratios reduced both the DNA release rate and the burst effect. The released DNA retained its structural and functional integrity. Intramuscular injection of PPE-EA-p43-LacZ complexes at N/P ratios of 0.5 and 1 resulted in enhanced beta-galactosidase expression in anterior tibialis muscle in Balb/c mice, as compared with naked DNA injections. Similarly, PPE-EA/IFN(alpha)2b DNA complexes generated an increased systemic level of interferon-alpha2b in mouse serum following intramuscular injection, as compared with naked DNA injection.     

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.     

DNA binding chelates for nonviral gene delivery imaging

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys(4)-Lys-(N-epsilon-[4-(psoralen-8-yloxy)]butyrate)-NH(2) has been characterized and labeled with reduced (99m)Tc pertechnetate (oxotechnetate). The psoralen moiety (a DNA binding group of PBC) allowed linking to double-stranded DNA upon short-term irradiation with the near UV range light (>320 nm). Approximately 30-40% of added (99m)Tc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of "naked" DNA and DNA complexes with lipid-based transfection reagents ("lipoplexes") has been performed after systemic or local administration of (99m)Tc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using (99m)Tc-PBC and (32)P-labeled DNA and lipoplexes. A markedly different biodistribution of (99m)Tc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. (99m)Tc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of "naked" (99m)Tc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, (99m)Tc PBC-DNA lipoplexes showed a slower elimination from the site of injection than (99m)Tc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.     

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

Delivery of genes to the lung has enormous potential in a wide variety of illnesses, from lung cancer to genetic deficiency diseases. Many delivery systems have been utilized, each with its own advantages and limitations. Polyethylenimine is a polycation capable of binding and compacting DNA, enabling intravascular plasmid delivery to normal tissues in such a way that the plasmid can be expressed in a proportion of the exposed cells. We have developed a novel intravenous method to deliver small amounts of plasmid to lung tissue, using nontoxic quantities of polyethylenimine in combination with albumin (or other soluble proteins). Injection of 1 microg or less of plasmid resulted in highly efficient gene expression in lung interstitial and endothelial tissues (0.5 to 1 ng luciferase per microg plasmid DNA), while larger quantities of plasmid reduced relative gene expression. Using luciferase as a reporter gene, single injections had maximal gene expression between 24 and 48 h, with a rapid decline thereafter. In contrast to some other delivery systems, however, no inhibition of gene expression occurred during multiple rounds of plasmid administration through 20 days. As a result, this method may have useful applications in diseases that could benefit from recurrent therapeutic gene delivery.     

Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes.

Polyethylenimine (PEI) has been studied as an efficient nonviral gene transfer vector. Here, we report the biodistribution fates and safety of plasmid DNA intravenously administered in PEI complexes. Using pCMVbeta as a model gene, the biodistribution of plasmid DNA was measured by quantitative polymerase chain reaction. A deletion mutant of pCMVbeta was used as an internal standard. After intravenous administration of PEI/DNA complexes, the serum levels of DNA rapidly declined for up to 15 min. However, after this point, the serum levels of DNA diminished slowly. At 15 min after dose, PEI/DNA complexes showed 33-fold higher distribution of DNA in the lung than did naked DNA. At 24 h, all the organs tested showed much higher levels of plasmid DNA in PEI/DNA complexes, with distribution in the liver and lung being three orders of magnitude higher than naked DNA. The mRNA expression of DNA was observed in various organs of PEI/DNA-treated mice at 12 days after dose. Once a week dosing of PEI/DNA complexes over 3 consecutive weeks did not alter the histology of the organs. However, twice a week dosing over 3 weeks induced a sign of inflammation in the liver. These results indicate that PEI enhances the delivery and retention of plasmid DNA in the organs, especially the liver, but that safe delivery requires proper dosing intervals.