Activation of NF-kB mediates ICAM-1 induction in respiratory cells exposed to an adenovirus-derived vector.

Gene transfer to the respiratory tract by replication-deficient adenoviruses is limited by the induction of inflammatory and immune responses. We previously demonstrated that a E1-E3-deleted recombinant adenovirus carrying the expression cassette for the cystic fibrosis gene (Ad.CFTR) upregulates the expression of the pro-inflammatory intercellular adhesion molecule-1 (ICAM-1) both in vitro and in vivo. In the present work we suggest a role for the nuclear factor-kB (NF-kB) in Ad.CFTR-dependent up-regulation of ICAM-1 in respiratory epithelial A549 cells. Specifically, Ad.CFTR induced translocation of NF-kB into the nucleus and binding to the proximal -228/-218 NF-kB consensus sequence on the ICAM-1 promoter. Ad.CFTR also stimulated a 13-fold increase in NF-kB-dependent expression of the CAT reporter gene under the control of a region of the ICAM-1 promoter, including the proximal NF-kB consensus sequence. The Ad.CFTR-dependent increase of ICAM-1 mRNA was abolished by inhibitors of NF-kB, such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, parthenolide and the synthetic peptide SN50. All these inhibitors abolished both Ad.CFTR-induced NF-kB DNA binding and transactivating activities. These results indicate a critical role of NF-kB in the pro-inflammatory response elicited by replication-deficient adenoviral vectors in respiratory cells.

Gene therapy for cystic fibrosis with aerosolized adenovirus-CFTR: characterization of the aerosol and scintigraphic determination of lung deposition in baboons.

For cystic fibrosis (CF) gene therapy using an aerosolized adenovirus expressing the CFTR gene, optimization of the inhalation conditions is a prerequisite to obtain sufficient amount of CFTR protein expression in the target areas of the respiratory tract. For such a purpose, in vivo radioisotopic imaging of the radiolabeled virus is a unique strategy for a quantitative assessment of the actual deposition. In the present study, an adenovirus CFTR (AdCFTR) was labeled with 99m Technetium gamma emitting isotope in such conditions that its bioactivity was preserved. The 99mTc-AdCFTR aerosol was characterized using both laser diffraction and cascade impaction for sizing with further determination of nebulized and inhalable fractions. After administration to baboons, scintigraphic quantitation of the regional lung distribution was performed and the actual dose deposited in the target area was estimated and expressed as an equivalent viral titer. Since a virus scintigraphy is not realistic in a hospital setting, we have developed an approach using 99mTc-DTPA (diethylene triamino pentaacetic acid) that could be used to predict the virus deposition. Indeed, similarities observed between 99mTc-DTPA and 99mTc-adenovirus aerosol imaging patterns validates the use of the 99mTc-DTPA scintigraphy that we propose as a pretherapeutic test for each patient prior to gene transfer.

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism.

Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CAR-binding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu, Tyr491Asp, Ala494Asp and Ala503Asp did not prevent the incorporation of trimeric fibers in the viral capsid but led to loss of CAR binding in vitro. Infectivity of the mutant viruses could be restored in vitro by introducing a ligand at the C-terminal end of the knob, confirming that the reduced infectivity of the fiber-modified virus was due to an impaired interaction of the viral particle with the CAR receptor. However, after systemic delivery, the in vivo biodistribution of impaired CAR-binding viruses without addition of a specific ligand was not altered when compared with wild-type Ad.

Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.

Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

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

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

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice.

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.

Selective in vivo transfection of murine biliary epithelia using polycation-enhanced adenovirus.

We have investigated the use of polycations to increase adenovirus-mediated expression of transgenic protein to the biliary epithelia with a view to gene therapy for hepatobiliary disease in CF. We have shown that adenovirus carrying the beta-galactosidase transgene transfect both human and mouse biliary epithelia in primary culture and that in both instances adenovirus transfection can be significantly increased by co-complexing with polycation. In vivo administration of 1 x 109 p.f.u. adenovirus co-complexed with the polyamine polyethyenimine (PEI) into the mouse biliary duct leads to >80% positively stained biliary epithelia while adenovirus alone at the same titre infected <5% biliary epithelia. We suggest that the use of low titre polycation enhanced adenoviral delivery to the biliary tree of CF patients could be of therapeutic significance. As a prelude to an extensive in vivo functional investigation in CF null mice we have shown that Ad5/polycation complexes deliver functional CFTR to non-CFTR expressing cells in vitro more efficiently than Ad5 alone.

Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers.

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.

Differential influence of the E4 adenoviral genes on viral and cellular promoters.

BACKGROUND: Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo. METHODS: Pairs of isogenic E1o and E1oE4o vectors were generated and compared. Levels of transgene expression were determined by Northern blot, ELISA and FACS analysis. Initiation of transcription was studied by nuclear run-on assays. RESULTS: Similar to the viral CMV and RSV promoters, the activity of the ubiquitous cellular PGK promoter required the presence of the E4 genes in vitro and in vivo. In contrast, transgene expression from selected liver- and tumor-specific promoters did not require E4 functions. CONCLUSION: Together with the reported low liver toxicity of E1oE4o vectors, the independence of E4 of liver-specific promoters renders such vectors interesting alternatives to the use of gutless vectors.

Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.

Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Effect of liposome-encapsulated clodronate pretreatment on synthetic vector-mediated gene expression in mice.

One of the main limitations for the use of synthetic vectors in gene therapy is their relatively low in vivo efficiency when compared with viral vectors. Here, we describe a pretreatment protocol with liposome-encapsulated clodronate in mice by which gene expression levels of a luciferase reporter gene could be increased up to nine-fold in the lung, after intravenous (i.v.) injection of glycerolipoplexes. Optimal results were obtained if mice were pretreated with liposome-encapsulated clodronate 1 day before injection of lipoplexes. The enhancement effect could be observed for lipoplexes prepared with different multivalent cationic glycerolipids. Most remarkably, polyplexes behaved in the opposite way. Liposome-encapsulated clodronate pretreatment strongly reduced reporter gene expression after i.v. injection of polyethylenimine-polyplexes (ExGen500).

Successful expression of beta-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein.

In utero somatic gene therapy in the later stages of pregnancy may allow targeting of organ systems which are difficult to reach later in life and to prevent the development of tissue damage otherwise caused by the early onset of inherited diseases. We report here on the percutaneous delivery of two adenoviral vectors, containing the beta-galactosidase reporter gene and the human Factor IX gene respectively, to the fetal liver and circulation by ultrasound-guided umbilical vein puncture similar to procedures used in human pregnancy. Vector spread, as detected by PCR analysis for the beta-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the beta-galactosidase transgene was detected in many fetal tissues by RT-PCR. High beta-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30percent of the hepatocytes stained positive, and in the adrenal cortex. Production of factor IX was determined by ELISA in the plasma of treated fetuses and newborn lambs and reached at birth up to 80percent of the normal human plasma concentration. This demonstrates a very hopeful proof of principle for the development of prenatal treatment of many genetic diseases but also requires more detailed investigations with respect to the observed systemic spread of the vector.

In vitro and in vivo effects of glucocorticoids on gene transfer to skeletal muscle.

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.