Toxicology study assessing efficacy and safety of repeated administration of lipid/DNA complexes to mouse lung.

For gene therapy to improve lung function in cystic fibrosis (CF) subjects, repeated administration of the gene transfer agent over the lifetime of patients is likely to be necessary. This requirement limits the utility of adenoviral and adeno-associated viral vectors (both previously evaluated in CF gene therapy trials) because of induced adaptive immune responses that render repeated dosing ineffective. For CF gene therapy trials, non-viral vectors are currently the only viable option. We previously showed that the cationic lipid formulation GL67A is the most efficient of several non-viral vectors analysed for airway gene transfer. Here, we assessed the efficacy and safety of administering 12 inhaled doses of GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA, into mice. We show that repeated administration of pGM169/GL67A to murine lungs is feasible, safe and achieves reproducible, dose-related and persistent gene expression (>140 days after each dose) using an aerosol generated by a clinically relevant nebuliser. This study supports progression into the first non-viral multidose lung trial in CF patients.

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung.

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models.

Previous studies with a mycobacterial heat shock protein (hsp-65) have demonstrated some efficacy using cationic liposome-mediated gene transfer in murine i.p. sarcoma models. To further analyze the efficacy of hsp-65 immunotherapy in clinically relevant models of localized cancer, immunocompetent mice bearing i.p. murine mesothelioma were treated with four i.p. doses of a cationic lipid complexed with plasmid DNA (pDNA) containing hsp65, LacZ, or a null plasmid. We observed >90% long-term survival (median survival, 150 days versus approximately 25 days, treated versus saline control, respectively) in a syngeneic, i.p. murine mesothelioma model (AC29). Long-term survivors were observed in all groups treated with lipid complexed with any pDNA. Lipid alone or DNA alone provided no demonstrable survival advantage. In a more aggressive i.p. model of mesothelioma (AB12), we observed >40% long-term survival in groups treated with lipid:pDNA complexes, again irrespective of the transgene. To ask whether these antitumor effects had led to an adaptive immune response against the tumor cell, we rechallenged long-term survivors in both murine models s.c. with the parental tumor cell line. Specific, long-lasting systemic immunity against the tumor was readily demonstrated in both models (AB12 and AC29). Consistent with these results, splenocytes from long-term survivors specifically lysed the parental tumor cell lines. Depleting the CD8+ T-cells from the splenocyte pool eliminated this lytic activity. Lipid:pDNA treatment of athymic, SCID, and SCID/Beige mice bearing a murine i.p. mesothelioma (AC29) resulted in only a slight survival advantage, but there were no long-term survivors. Treatment of immunocompetent mice depleted of specific immune effector cells demonstrated roles for CD8+ and natural killer cells. Although the exact mechanism(s) responsible for these antitumor effects is unclear, the results are consistent with roles for both innate and adaptive immune responses. An initial tumor cell killing stimulated by cationic lipid:pDNA complexes appears to be translated into long-term, systemic immunity against the tumor cell. These results are the first to demonstrate that adaptive immunity against a tumor cell can be induced by the administration of lipid:pDNA complexes. Multiple administrations of cationic lipid complexed with pDNA lacking an expressed transgene could provide a promising generalized immune-mediated modality for treating cancer.

Fusion of Sindbis virus with model membranes containing phosphatidylethanolamine: implications for protein-induced membrane fusion.

The pH-induced fusion of Sindbis virus with model lipid membranes containing phosphatidylethanolamine has been studied using a quantitative fluorescence technique. The headgroup and acyl chain domains of the lipids have been altered systematically to determine their effect on fusion. Unsaturated phosphatidylethanolamines (PE) have been found to promote fusion, either by themselves, or in combination with phosphatidylcholines (PC). Cholesterol added to a mixture of unsaturated PE and PC was also shown to increase the extent of viral fusion. The results of these studies have been interpreted in terms of a tentative model for the molecular aspects of the target membrane which are necessary for viral fusion. In this model, the target membrane must have a sufficiently-sized domain containing poorly hydrated lipids which are capable of existing in a non-bilayer arrangement.

Biophysical characterization of cationic lipid: DNA complexes.

To better understand the structures formed by the interaction of cationic lipids with DNA, we undertook a systematic analysis to determine the biophysical characteristics of cationic lipid:DNA complexes. Four model cationic lipids with different net cationic charge were found to interact in similar ways with DNA when that interaction was compared in terms of the apparent molar charge ratio of lipid to DNA. When DNA was present in charge excess over the cationic lipid, the complex carried a net negative charge as determined by zeta potential measurements. Under these conditions, some DNA was accessible to ethidium bromide, and free DNA was observed in agarose gels and in dextran density gradients. Between a lipid:DNA charge ratio of 1.25 and 1.5:1, all the DNA became complexed to cationic lipid, as evidenced by its inaccessibility to EtBr and its complete association with lipid upon agarose gel electrophoresis and density gradient separations. These complexes carried a net positive charge. The transition between negatively and positively charged complexes a occurred over a very small range of lipid to DNA ratios. Employing a fluorescent lipid probe, the addition of DNA was shown to induce lipid mixing between cationic lipid-containing vesicles. The extent of DNA-induced lipid mixing reached a maximum at a charge ratio of about 1.5:1, the point at which all the DNA was involved in a complex and the complex became positively charged. Together with freeze-fracture electron micrographs of the complexes, these biophysical data have been interpreted in light of the existing models of cationic lipid:DNA complexes.

The in vivo effects of rhIL-1 alpha therapy on human monocyte activity.

Pleiotropic cytokines such as interleukin-1 alpha (IL-1 alpha) have multiple effects on peripheral blood monocytes (PBMs). This study examined the ability of in vivo recombinant human IL-1 alpha (rhIL-1 alpha) therapy to enhance clinically important monocyte functions in ovarian cancer patients prior to chemotherapy. After 4 days of continuous infusion, in vivo rhIL-1 alpha therapy amplified both the number and activity of PBMs. Therapy with rhIL-1 alpha increased the number of PBMs sixfold. These monocytes had a significantly increased ability to produce superoxide anion in response to phorbol 12,13-dibutyrate stimulation. Their ability to secrete spontaneously the immunomodulatory cytokines IL-1 alpha and IL-1 beta was significantly increased, but their ability to secrete tumor necrosis factor alpha (TNF-alpha) was not significantly elevated. These effects of rhIL-1 alpha infusion on cytokine secretion by PBMs appear to be related to rhIL-1 alpha-induced increases in the mRNA levels for these cytokines. In contrast, rhIL-1 alpha therapy did not significantly alter PBM response to lipopolysaccharide (10 micrograms/ml). In summary, infused rhIL-1 alpha, in addition to its use as a myeloprotective agent, has enhancing effects on the number and activity of PBMs. The effects of rhIL-1 alpha infusion on PBM function demonstrated here should at least transiently increase the ability of monocytes to combat infection and enhance host immune response.

Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes.

We have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mM NaCL as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitro aerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (1-3 microns). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.

Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice.

A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-gamma, TNF-alpha, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-alpha were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.

EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo.

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.

Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice.

The potential of cationic liposomes as nonviral vectors for in vivo gene delivery to the liver and to intrahepatic hepatocellular carcinoma (HCC) was investigated. Mice were injected via the tail vein or portal vein with a cationic lipid complexed to plasmid DNA (pDNA) encoding the chloramphenicol acetyltransferase (CAT) reporter gene at various cationic lipid:pDNA molar ratios to analyze the efficiency of gene delivery after intravenous administration. Tail vein injection resulted in high CAT expression levels in lung and spleen and low levels in the liver. Portal vein injection, by comparison, significantly enhanced hepatic reporter gene expression but also resulted in pronounced hepatic toxicity. Gene delivery to intrahepatic tumors produced by intrahepatic injection of human HCC cells was analyzed in nude mice. Tail vein injection as well as portal vein injection resulted in low levels of gene expression in intrahepatic tumors. By comparison, high levels of gene expression were achieved by direct, intratumoral injection of liposome-pDNA complexes, with only minimal expression in the surrounding normal liver. Therefore, direct liposome-pDNA complex injection appears far superior to systemic or portal intravenous administration for gene therapy of localized intrahepatic tumors, and may be a useful adjunct in the treatment of human HCCs.

Aerosolization of cationic lipid:pDNA complexes--in vitro optimization of nebulizer parameters for human clinical studies.

Previously, we have described the optimization of the aerosol delivery of a nonviral gene therapy vector to the lungs of rodents (Eastman et al., 1997b). Although aerosolizing cationic lipid:pDNA complexes into a whole-body exposure chamber resulted in high levels of reporter gene expression in the lungs of BALB/c mice, the conditions employed were not optimal for the delivery of lipid:pDNA complexes to the lungs of human patients. That is, the consumption rate of the material in the nebulizer, and thus the delivery time, were very slow and the aerosol was delivered in a continuous flow. Here we describe in vitro experiments used to develop a cationic lipid:pDNA aerosol with characteristics more suitable for delivery to the lungs of humans, as a necessary prerequisite for conducting a clinical study with human cystic fibrosis patients. Using cascade impactors and all-glass impingers, we have screened several commercially available nebulizers for their ability to deliver intact, respirable, active lipid:pDNA complexes in the shortest time possible, and have identified the Pari LC Jet Plus nebulizer as the optimal nebulizer that meets these criteria. Using this nebulizer in an intermittent mode to mimic breath actuation, consumption rates of approximately 0.6 ml/min of the cationic lipid:pDNA complexes (6 mM cationic lipid:8 mM pDNA) were obtained. The plasmid DNA remained intact and the complexes were shown to maintain activity throughout the nebulization run. Based on measurements of the nebulized dose and the mass median aerodynamic diameter, we calculate a delivered dose of approximately 22 micromol (7.2 mg) of pDNA for each 8 ml of cationic lipid:pDNA complex aerosolized to the lungs of a human patient. This dose should be sufficient to test the clinical efficacy of cationic lipid-mediated gene delivery for the treatment of cystic fibrosis.

Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells.

To better understand the barriers associated with cationic lipid-mediated gene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filter supports at an air-liquid interface were used to study the binding and uptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of binding and uptake of cationic lipid:pDNA complexes by these cell systems were monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiated from internalized probe by adding trypan blue, which quenched the fluorescence of bound but not internalized probes. For proliferating cells, binding and internalization of the cationic lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the complexes was observed using polarized epithelial cells. However, after aspirating a small area of cells from the filter support, virtually all of the cells adjoining this newly formed edge bound and internalized the cationic lipid:pDNA complexes. To determine if their uptake in edge cells was related to the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies known to disrupt tight junctions. Cells treated in this manner bound and internalized cationic lipid:pDNA complexes efficiently and also expressed significant levels of transgene product. Control cells with intact tight junctions neither bound complexes nor expressed significant transgene product. These data confirm and extend earlier observations that the polarized apical membranes of airway epithelial cells are resistant to transfection by lipid:pDNA complexes. Further, in contrast to previous studies that have shown the entry step of complexes is not an important barrier for COS and HeLa cells, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tight junctions of polarized epithelial cells may improve gene delivery to these cells for diseases such as cystic fibrosis (CF).

Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

A concentrated and stable aerosol formulation of cationic lipid:DNA complexes giving high-level gene expression in mouse lung.

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

The role of CpG motifs in immunostimulation and gene therapy.

The mammalian immune system has evolved mechanisms to recognize and respond to 'danger' signals arising from pathogens. Among those danger signals are the unmethylated CpG dinucleotide motifs found in bacteria. At least some of the recognition of these sequences is through cellular components of the innate immune system, such as macrophages. Cytokines released by these cells in response to CpG motifs in turn activate other immune cells, such as NK cells and T cells, and can drive the development of adaptive immune responses. These proinflammatory, Th1 responses can also be generated intentionally with small oligodeoxynucleotides containing stimulatory CpG motifs, and have beneficial properties as vaccine adjuvants and in cancer immunotherapy. These proinflammatory responses have also been seen in gene therapy applications, especially in systemic delivery systems in which plasmid DNA vectors have been introduced with a vehicle such as a cationic lipid. For many gene therapy applications, finding ways to counter the immunostimulatory properties of plasmid DNA vectors is an important approach designed to enhance the vector safety profile, thereby increasing its effective therapeutic index.

Toxicity of cationic liposome-DNA complex in lung isografts.

BACKGROUND: Cationic lipids have been successfully employed as vectors for gene transfer in lung grafts, yet those lipid vectors have potential toxicity. Furthermore, the optimal concentration of cationic lipids for gene transfection to lung grafts has not been determined. We evaluated liposome concentration/toxicity relationships in an in vivo rat lung transplantation model. METHODS: Left lungs were harvested and infused via the pulmonary artery with chloramphenicol acetyl-transferase (CAT)-DNA/lipid 67 (cationic lipid)/dioleoylphosphatidylethanolamine complex (4:1:2 in a final concentration ratio). Donor lungs were allocated into six groups according to lipid 67 concentration: group 1, 0 microM (control); group 2, 10 microM; group 3, 50 microM; group 4, 100 microM; group 5, 250 microM; group 6, 500 microM. Forty-eight hours after orthotopic transplantation, the recipient contralateral right main pulmonary artery and bronchus were ligated. The graft was ventilated with 100% oxygen for 5 min. Arterial blood gas analysis (PaO2, PaCO2), peak airway pressure (PAP), and CAT activity of the grafts were measured. RESULTS: Recipient survival, and PaO2, PAP, and CAT levels correlated with the lipid-DNA complex concentration. The grafts in groups 4-6 were more injured as evidenced by decreased PaO2 and increased PAP levels in comparison to the control group. CAT level was significantly lower in group 2 than in groups 3-6. CONCLUSIONS: The pulmonary toxicity of cationic lipid is dose-dependent. The balance between lung graft function and transgene expression is optimal at a lipid 67 concentration of 50 microM.

Successful in vivo and ex vivo transfection of pulmonary artery segments in lung isografts.

OBJECTIVE: Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Efficient gene transfection to the whole organ may prove problematic. Proximal pulmonary artery endothelial transfection might provide beneficial downstream effects on the whole graft. The aim of this study was to determine the feasibility of transfecting proximal pulmonary artery segments in lung isografts. METHODS: Male Fischer rats were divided into six groups. In vivo transfection: In group I (n = 7), a proximal segment of the left pulmonary artery was isolated and injected with saline solution by means of a catheter inserted through the right ventricle. After an exposure period of 20 minutes, clamps were removed and blood flow was restored. In group II (n = 7), the isolated arterial segments were injected with adenovirus carrying the Escherichia coli LacZ gene encoding for beta-galactosidase. Ex vivo transfection: In group III (n = 5), arterial segments were injected ex vivo with saline solution and in group IV (n = 5) with the adenovirus construct. In group V (n = 6), arteries were injected with saline solution and in group VI (n = 11) with liposome chloramphenicol acetyl transferase cDNA. In groups I to IV, animals were killed on postoperative day 3 and transgene expression was assessed by Bluo-Gal staining. In groups V and VI, animals were killed on postoperative day 2 and transgene expression was assessed by chloramphenicol acetyl transferase activity assay. RESULTS: Transgene expression was detected grossly and microscopically in endothelial and smooth muscle cells of pulmonary artery segments from all surviving animals of groups II and IV. In group VI, chloramphenicol acetyl transferase activity was significant in all assessed arterial segments. CONCLUSION: Significant transgene expression is observed in proximal pulmonary artery segments after both in vivo and ex vivo exposure.