Levels Of Luciferase Gene Expression Research Articles

Dendrimer-Coated Gold Nanoparticles for Efficient Folate-Targeted mRNA Delivery In Vitro.

Messenger RNA (mRNA) is not an attractive candidate for gene therapy due to its instability and has therefore received little attention. Recent studies show the advantage of mRNA over DNA, especially in cancer immunotherapy and vaccine development. This study aimed to formulate folic-acid-(FA)-modified, poly-amidoamine-generation-5 (PAMAM G5D)-grafted gold nanoparticles (AuNPs) and to evaluate their cytotoxicity and transgene expression using the luciferase reporter gene (FLuc-mRNA) in vitro. Nanocomplexes were spherical and of favorable size. Nanocomplexes at optimum nanoparticle:mRNA (w/w) binding ratios showed good protection of the bound mRNA against nucleases and were well tolerated in all cell lines. Transgene expression was significantly (p < 0.0001) higher with FA-targeted, dendrimer-grafted AuNPs (Au:G5D:FA) in FA receptors overexpressing MCF-7 and KB cells compared to the G5D and G5D:FA NPs, decreasing significantly (p < 0.01) in the presence of excess competing FA ligand, which confirmed nanocomplex uptake via receptor mediation. Overall, transgene expression of the Au:G5D and Au:G5D:FA nanocomplexes exceeded that of G5D and G5D:FA nanocomplexes, indicating the pivotal role played by the inclusion of the AuNP delivery system. The favorable properties imparted by the AuNPs potentiated an increased level of luciferase gene expression.

Synthesis and characterization of layered double hydroxides and their potential as nonviral gene delivery vehicles.

Layered double hydroxides (LDHs) exhibit characteristic anion-exchange chemistry making them ideal carriers of negatively charged molecules like deoxyribonucleic acid (DNA). In this study, hydrotalcite (Mg−Al) and hydrotalcite-like compounds (Mg−Fe, Zn−Al, and Zn−Fe), also known as LDHs, were evaluated for their potential application as a carrier of DNA. LDHs were prepared by coprecipitation at low supersaturation and characterized by Powder X-ray diffraction (XRD), infrared (IR), Raman, and inductively coupled plasma—optical emission spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). XRD patterns showed strong and sharp diffraction peaks for the (003) and (006) planes indicating well-ordered crystalline materials. TEM images yielded irregular circular to hexagonal-shaped particles of 50–250 nm in size. Varying degrees of DNA binding was observed for all the compounds, and nuclease digestion studies revealed that the LDHs afford some degree of protection to the bound DNA. Minimal toxicity was observed in human embryonic kidney (HEK293), cervical cancer (HeLa) and hepatocellular carcinoma (HepG2) cell lines with most showing a cell viability in excess of 80 %. All LDH complexes promoted significant levels of luciferase gene expression, with the DNA:Mg−Al LDHs proving to be the most efficient in all cell lines.

In vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma

Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin–conjugated HVJ envelope vector

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer.

177. A Novel Class of Cationic Amphiphiles for Airway Gene Delivery

A new class of cationic amphiphiles was synthesized and characterized for their transfection activity in mouse lung by intratracheal delivery. These cationic compounds, consisting of a backbone of the bis(2-hydroxy-3-alkiammonio)propyl methylamine with various alkyl chains (C12 to C18) linked to two amine groups, undergo temperature and concentration dependent transition from liposomes to micelles. They are highly soluble in water and readily form complexes with DNA, depending on the chain length of the alky group in the compounds. We compared luciferase gene expression in lungs of CD-1 mice that received 25 mg compacted pCMV-Luc plasmid using the cationic amphiphiles in 30 ml water via intratracheal or intranasal administration. Intratracheal administration of pCMV- Luc complexed with the cationic amphiphiles resulted in various levels of luciferase gene expression in the lung 24hr post gene delivery. Transfection activity is primarily determined by the structure of the compounds, less by the N/P ratio. Compounds exhibited an optimal transfection activity in the lung are different from those exhibiting the highest activity in A549 lung epithelial cells. While transfection activity is comparable to standard transfection reagent such as DOTAP/Cholesterol liposomes in cell culture, the activity of the cationic amphiphiles with optimal structure is 50–100 folds higher for intratracheal transfection in the lung. These results demonstrated that these new amphiphiles are promising carriers for lung gene therapy. The study was supported by NIH grant RO1 HL075542. Key Words: airway gene delivery, cationic amphiphies, gene therapy, intratracheal administration

692. Aerosol Delivery of Concentrated pDNA/ PEI Formulations to the Murine Lung

Plasmid DNA (pDNA) complexed to the cationic polymer polyethyleneimine (PEI) has been shown to retain transfection efficiency following aerosolisation and as a result, PEI has become a popular choice of gene transfer agent for lung gene therapy. Development of PEI for clinical applications has been hampered by the relatively low pDNA/PEI concentrations that can be prepared in the laboratory - typically less than 0.4mg/ml. We have investigated the use of a large-scale ultrafiltration protocol to generate stable concentrated pDNA/PEI formulations in excess of 8mg/ml and we have tested these formulations in a mouse lung model. Plasmid pCIKLux expressing the firefly luciferase gene under the control of the cytomegalovirus immediate/early promoter was complexed with branched 25kDa PEI (N:P of 10:1) at an initial concentration of 0.2mg/ml. This formulation was concentrated using a pressurised ultrafiltration cell incorporating a 100kDa cut-off cellulose membrane to produce final formulations containing 1,2,4 and 8mg/ml pDNA/ PEI complexes. Following concentration, pCIKLux/PEI formulations were administered to the lungs of female BALB/c mice (8|[ndash]|16wks) via nasal instillation (100|[mu]|l) or whole body aerosol exposure (10ml aerosol) and luciferase expression was determined in whole lung homogenates 24hrs later. Although instillation of pCIKLux/PEI formulations at 1mg/ml resulted in good levels of luciferase gene expression (71.1|[plusmn]|16.7 RLU/mg protein), the observed expression was not significantly different from that achieved following instillation of standard 0.2mg/ml formulation (41.6|[plusmn]|10.4 RLU/mg) (p=0.35 ANOVA). In addition, instillation of complexes to the mouse lung was associated with considerable dose-dependent toxicity, manifested by weight loss, histological changes and elevated neutrophil levels in bronchoalveolar lavage fluid (BALF). Instillation of pDNA/PEI complexes at 1mg/ml resulted in significant mortality and higher concentrations were not assessed. In contrast, aerosol delivery of concentrated pDNA/PEI formulations to the mouse lung resulted in high levels of reporter gene expression with minimal associated lung toxicity. Aerosol delivery of pCIKLux/PEI at 1mg/ ml produced luciferase expression at 133.2|[plusmn]|11.2 RLU/mg compared to 18.8|[plusmn]|1.9 RLU/mg for standard 0.2mg/ml formulations (p=0.0003 ANOVA). Higher concentrations resulted in increased luciferase expression at 2mg/ml (215.5|[plusmn]|18.9 RLU/mg), 4mg/ml (274.0|[plusmn]|39.2 RLU/mg) and even 8mg/ml (310.3|[plusmn]|55.5 RLU/mg) although expression did not increase in a linear fashion relative to the delivered dose. Minimal evidence of lung toxicity was observed following aerosol delivery with moderate weight loss and a slight elevation of BALF neutrophil numbers observed only at doses of 4mg/ml and above.

Comparative Efficacy of Intratracheal Adeno-Associated Virus Administration to Newborn Rats

Transient local overexpression of genes that promote lung defense or repair may help to protect or promote alveolar development in premature neonates. We showed that the use of adenoviral vectors in neonates was limited by the induction of lung growth disorders. In the present work we compare the efficiency of gene transfer to the neonatal lung by three adeno-associated viral vectors: rAAV1, rAAV2, and rAAV5. Transduction efficiency was first measured in vitro, by infecting A549 immortalized human lung epithelial cells, and primary epithelial and mesenchymal cells isolated from human fetal lung. AAV vectors yielded similar low levels of luciferase gene expression in the different cell types. In vivo transduction efficiency was evaluated in newborn rats, with AAV-LacZ vectors being intratracheally instilled at 3 days of age. Both rAAV5 and rAAV1, but not rAAV2, induced significant lung beta-galactosidase expression, which persisted on day 35. Highest beta- galactosidase levels were measured with rAAV5, but remained far lower than those obtained with adenoviral vectors. A transient increase in alveolar macrophages was observed on day 6, but not on day 8, after rAAV5-LacZ instillation. Morphometric evaluation of lung structures was performed on day 21, and showed no altered lung growth. We conclude that rAAV1 or rAAV5 was more efficient at mediating gene transfer in the neonatal lung than was rAAV2, without adversely affecting lung development. However, in vivo transgene expression was relatively low, and needs to be improved for future therapeutic use of these adeno-associated vectors.

Structure–function relationships of gene delivery vectors in a limited polycation library

A library of 13 polylysine- graft-imidazoleacetic acid conjugates was synthesized to examine the collective effects of polymer molecular weight, side chain substitution, and DNA:polymer ratio on cytotoxicity, transfection efficiency, and polycation–DNA interaction. In general, the relationships between the physicochemical characteristics and the gene transfer capabilities of these polycations appear nonlinear. The in vitro cytotoxicity of these polymers decreased, while total protein expression increased, with decreasing molecular weight and increasing imidazole content. Flow cytometry experiments indicated, however, that an increase in marker gene expression does not always correlate with the total number of cells transfected, even when similar polymer structures are used for transfection. The maximum level of luciferase gene expression was mediated by transfection with a low molecular weight, high imidazole content (9400 M w, 95 mol% imidazole) polymer. The extent of DNA condensation, as determined by ethidium bromide fluorescence quenching, also decreased with decreasing polymer molecular weight and increasing imidazole content. Relative binding affinity between DNA and the polycations, measured via competitive binding in the presence of a synthetic polyanion, decreased with decreasing polymer molecular weight; however, the relative affinity also appeared to increase with increasing imidazole, suggesting that electrostatic contributions are not solely responsible for DNA–polycation binding interactions. This limited library and corresponding structure/function analysis forms the foundation upon which larger, more comprehensive polycationic libraries can be designed and evaluated to further understand how polycation transfection reagents function.

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines.

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/−) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.

The IDDM-associated solitary retroviral promoters DQ-LTR3 and DQ-LTR13 have a distinct impact on the expression of selected DQB1 genes in different cell lines in vitro.

The HLA complex on chromosome 6, especially of the HLA class II genes, plays an important role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). Three solitary long terminal repeats (LTRs) have been described in the vicinity of HLA DQ, two of them modifying the genetic susceptibility to type 1 diabetes on high-risk HLA DQ haplotypes. Therefore, we have investigated the putative regulatory properties of these retroviral promoters in different cell lines, including a number of B- and T-lymphoblastoid cell lines bearing different DQ haplotypes, employing a transient reporter gene assay. The transcriptional activity of appropriate constructs harboring an LTR linked to the luciferase reporter gene revealed different expression patterns which varied considerably between the investigated cell lines. DQ-LTR3 showed clear activities, whereby the levels of luciferase gene expression were also increased approximately 200-fold in the teratocarcinoma cell line GH. For the different B- and T-cell lines, no significant activity was detected for any of the investigated LTRs. Furthermore, we have analyzed the effect of DQ-LTR13 on distinct DQB1 promoters and could show an increased activity of the DQB1*0302 promoter under the influence of DQ-LTR13. It varied from 1.5- to 6-fold in different cell lines depending on the transcriptional orientation and position of the LTR and was independent of DQ-LTR3. Moreover, the impact of the LTR was different for the DQB1*0201 promoter demonstrating a decreasing effect. These data indicate that a DQ-LTR13-mediated impact on the DQB1 promoter activity exists which differs clearly between selected promoters.

Global non-viral gene transfer to the primate brain following intravenous administration.

Expression plasmids encoding either luciferase or beta-galactosidase were encapsulated in the interior of an "artificial virus" comprised of an 85 nm pegylated immunoliposome, which was targeted to the rhesus monkey brain in vivo with a monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb enables the liposome carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across the neuronal plasma membrane following intravenous injection. The level of luciferase gene expression in the brain was 50-fold higher in the rhesus monkey as compared to the rat. Widespread neuronal expression of the beta-galactosidase gene in primate brain was demonstrated by both histochemistry and confocal microscopy. This approach makes feasible reversible adult transgenics in 24 hours.

Marked enhancement in gene expression by targeting the human insulin receptor.

Exogenous genes can be delivered to cells without viral vectors using an "artificial virus" comprised of nonviral plasmid DNA encapsulated in the interior of 85 nm pegylated immunoliposomes (PIL). The liposomes are targeted to cells with receptor-specific targeting ligands such as receptor-specific peptidomimetic monoclonal antibodies. The levels of luciferase gene expression in human or rat glioma cells are measured after targeting the PIL-encapsulated plasmid DNA via the human insulin receptor, the human epidermal growth factor receptor, or the rat transferrin receptor. The luciferase expression plasmids were either derived from pCEP4, which contains the Epstein-Barr nuclear antigen-1/oriP replication system, or from pGL2, which lacks this system for episomal replication of plasmid DNA. Depending on the plasmid construct used and the receptor targeted, the peak luciferase gene expression varied more than 200-fold from 1.8 +/- 0.1 to 419 +/- 31 pg luciferase per mg cell protein. With the same plasmid, the peak level of gene expression following delivery to the cell via the human insulin receptor was 100-200-fold higher than gene expression following delivery via either the epidermal growth factor receptor or the transferrin receptor. There was no gene expression if the targeting ligand on the PIL was replaced with a nonspecific isotype control antibody. The extent to which an exogenous gene is expressed within a cell via a nonviral, receptor-mediated gene transfer technology is determined by the receptor specificity of the targeting ligand. The highest levels of gene expression are obtained after targeting the insulin receptor, and this may derive from the nuclear targeting properties of this receptor system.

Receptor-mediated delivery of an antisense gene to human brain cancer cells.

The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High-grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR-dependent gliomas. A human EGFR antisense gene driven by the SV40 promoter in a non-viral plasmid carrying elements that facilitate extra-chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83-14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR). Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein-labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on beta-galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [(3)H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR. The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70-80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors.

In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers

Gene transfer into the airways could be of importance for the treatment of chronic lung diseases such as cystic fibrosis. In the past few years several attempts have been made to effectively deliver DNA to the lung using different viral and non-viral vector systems. Viral vectors and cationic lipids have been tested intensively but the properties of cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to deliver DNA to the airways have not been studied. Surfactant preparations have been shown to influence pulmonary adenoviral and naked plasmid DNA mediated gene transfer in vivo. We investigated the gene delivery efficiency of branched PEI 25 kDa and fractured dendrimers to the murine lung in vivo and also examined the effect of surfactant on PEI 25 kDa mediated gene transfer to the lung. Cationic polymer/DNA complexes were prepared in 25 mM HEPES buffer (pH = 7.4) or double distilled water and administered to the lungs of BALB/c mice via cannula intubation. The trachea, left and right lung, heart, liver and esophagus were examined for luciferase activity. Inflammation was assessed by performing standard histology. PEI/DNA complexes showed a high level of luciferase gene expression in the lung. Complexes formed in double distilled water exhibited higher gene expression than complexes formed in 25 mM HEPES buffer (pH 7.4). The optimal N/P ratio was found to be N/P = 10 in double distilled water. Luciferase activity was only detected in the lung and decreased rapidly in a time-dependent manner. The addition of a natural surfactant preparation, Alveofact, slightly reduced gene transfer of branched PEI 25 kDa. Luciferase gene expression obtained by using fractured dendrimers was very low. The present study demonstrates that PEI 25 kDa, but not polyamidoamine dendrimers, effectively mediates transient gene transfer to the murine lung after intratracheal intubation. In conclusion, branched PEI 25 kDa was found to be an effective vector for pulmonary gene delivery in vivo, being superior to fractured dendrimers.

Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA.

Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis.

Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty. The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes. The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.