Search

Lung delivery of plasmid DNA encoding the CFTRgene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTRgene therapy in patients with cystic fibrosis.

Acute lung injury is a common cause of morbidity and mortality following pulmonary or systemic infections. Surfactant protein-D is a member of the collectin family of proteins, which play important roles in innate host defense of the lung. In this study, the effect of exogenous recombinant human SP-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury was assessed.

Hydrodynamic delivery is an efficient and inexpensive procedure to deliver a wide range of nucleic acids to hepatic tissues and other organs in vivo. The successful application of hydrodynamic delivery is dependent on the rapid injection of a large aqueous volume containing DNA, RNA or other molecules into the vasculature of the liver. In this review, the development of the procedures for hydrodynamic delivery will be described and the parameters necessary for attaining maximal gene expression will be highlighted. A review of the mechanisms for transfecting hepatocytes, as well as potential uses of this approach in various research and clinical applications, will also be discussed.

Systemic delivery of synthetic gene transfer vectors such as cationic lipid:plasmid DNA (pDNA) complexes elicits a range of acute physiologic responses, which in the context of therapeutic gene delivery represent dose-limiting toxicities. The most prominent responses are transient leukopenia, thrombocytopenia, serum transaminase elevations, and elevations of proinflammatory cytokines such as interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α). The unmethylated CpG sequences present in plasmid DNA have been implicated as a major cause of the robust cytokine response that follows systemic administration of cationic lipid:pDNA complexes. However, the factors causing the additional significant toxicities (leukopenia, thrombocytopenia, and serum transaminase elevations) recently shown to be associated with vector administration have not been defined. We show here that DNA sequences, such as immune stimulatory CpG sequences, play a significant role in inducing the additional acute toxicities associated with cationic lipid:pDNA complex administration. Importantly, while methylating these CpG sequences results in greatly reduced cytokine levels, this modification does not eliminate their ability to generate the other systemic toxicities. Examples of non-CpG DNA sequences that induce distinct toxicity profiles when administered systemically in the form of cationic lipid:DNA complexes are also identified. Taken together, these results imply that specific DNA sequences are responsible for a significant portion of the systemic toxicities observed after administration of cationic lipid:pDNA complexes.

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.

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.

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-γ, TNF-α, 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-α 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.

Background:The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor–ß1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. Methods:The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor–ß1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. Results:On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor–ß1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor–ß1 sense construct compared with antisense controls (arterial oxygen tension = 411 ± 198 vs 103 ± 85 mm Hg, respectively; P= .002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 ± 0.5 vs 2.8 ± 0.6; P= .04; airway rejection scores: 1.3 ± 0.7 vs 2.3 ± 0.8, respectively; P= .02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. Conclusions:The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor–ß1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology. (J Thorac Cardiovasc Surg 2000;119:913-20)

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.

AbstractEnhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid : plasmid DN A (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanol-amine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphati-dylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid: pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.

Background. Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Proximal pulmonary artery endothelial transfection may provide beneficial downstream effects on the whole lung graft. We have already demonstrated the feasibility of in vivo and ex vivo transfection in proximal pulmonary artery segments of rat lung grafts. The aim of this study was to determine the optimal conditions for and duration of transfection.

Background. Proximal pulmonary artery segment (PPAS) endothelial transfection of lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection and may provide beneficial downstream effects on the whole lung graft. Transfection immediately after lung transplantation may be efficacious in ameliorating allograft dysfunction after transplantation.

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 Sss I-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 causeand-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-1 alpha 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.

ABSTRACTCationic 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 g 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 CFTRgene 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 CFTRin CF lungs is a viable and promising approach for treatment of the disease.

We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, α-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al.(Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R.(1992) Cell68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[α-32P]ATP with half-maximal binding at 25 and 50 μM, respectively. These values compare well with those reported for 8-N3-[α-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.

Pleiotropic cytokines such as interleukin‐lα (IL‐lα) have multiple effects on peripheral blood monocytes (PBMs). This study examined the ability of in vivo recombinant human IL‐lα (rhIL‐lα) therapy to enhance clinically important monocyte functions in ovarian cancer patients prior to chemotherapy. After 4 days of continuous infusion, in vivo rhIL‐lα therapy amplified both the number and activity of PBMs. Therapy with rhIL‐lα 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‐lα and IL‐1β was significantly increased, but their ability to secrete tumor necrosis factor a (TNF‐α) was not significantly elevated. These effects of rhIL‐lα infusion on cytokine secretion by PBMs appear to be related to rhlL‐lα‐in‐ duced increases in the mRNA levels for these cytokines. In contrast, rhIL‐lα therapy did not significantly alter PBM response to lipopolysaccharide (10 μg/ml). In summary, infused rhIL‐lα, in addition to its use as a myelo‐ protective agent, has enhancing effects on the number and activity of PBMs. The effects of rhIL‐lα infusion on PBM function demonstrated here should at least transiently increase the ability of monocytes to combat infection and enhance host immune response.

ABSTRACTPreviously, 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 vitroexperiments 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 mMcationic lipid:8 mMpDNA) 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 ~ 22 mol (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.

ABSTRACTWe 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 mMNaCl 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 vitroaerosolization 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 (13 m). 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.

ABSTRACTStudies 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-αα(TNF-αα), and interferon-γγ(IFN-γγ) 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.

ABSTRACTAdvances 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 mMpDNA) 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 vitroand 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.

Objective:Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. Methods:Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature:In group I (n= 4), grafts were stored for 4 hours at 23° C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n= 4), grafts were stored at 10° and 4° C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time:Grafts were preserved at 10° C for 1, 2, 3, 4 (n= 4), and 10 hours (n= 5). Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression:Grafts were preserved at 10° C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. Results:Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4° C than in those transfected at 10° and 23° C. Storage for 1 hour at 10° C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. Conclusion:Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.(J Thorac Cardiovasc Surg 1998:115;38-44)