Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia.

Developing gene therapy for cystic fibrosis has been hindered by limited binding and endocytosis of vectors by human airway epithelia. Here we show that the apical membrane of airway epithelia express the urokinase plasminogen activator receptor (uPAR). Urokinase plasminogen activator (uPA), or a 7-residue peptide derived from this protein (u7-peptide), bound the receptor and stimulated apical endocytosis. Both ligands enhanced gene transfer by nonspecifically bound adenovirus and adeno-associated virus vectors and by a modified adenovirus vector that had been coupled to the u7-peptide. These data provide the first evidence that targeting an apical receptor can circumvent the two most important barriers to gene transfer in airway epithelia. Thus, the uPA/uPAR system may offer significant advantages for delivering genes and other pharmaceuticals to airway epithelia.

Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice.

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV).

BACKGROUND: Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates. METHODS: The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography. RESULTS: Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice. CONCLUSIONS: This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate.

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.

Characterization of the oligosaccharide structures associated with the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane-associated glycoprotein. The protein can exist in three different molecular weight forms of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively. The most common mutation in cystic fibrosis (CF) results in the synthesis of a variant (DeltaF508-CFTR) that is incompletely glycosylated and defective in its trafficking to the cell surface. In this study, we have analyzed the oligosaccharide structures associated with the different forms of recombinant CFTR, by expressing and purifying the channel protein from either mammalian Chinese hamster ovary (CHO) or insect Sf9 cells. Using glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) we determined that purified CHO-CFTR contained polylactosaminoglycan (PL) sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. The presence of PL sequences on the recombinant CHO-CFTR is consistent with a normal feature of mammalian processing, since endogenous CFTR isolated from T84 cells displayed a similar pattern of glycosylation. The present study also reports on the use of FACE for the qualitative analysis of small amounts of glycoprotein oligosaccharides released enzymatically.

cAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes.

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl(-) currents in either symmetrical MgCl(2) or NaCl. ATP, adenosine 5'-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl(-) and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Delivery of purified, functional CFTR to epithelial cells in vitro using influenza hemagglutinin.

To assess the feasibility of protein replacement as a potential therapy for cystic fibrosis, we have evaluated the ability of influenza hemagglutinin (HA) to mediate the delivery of purified cystic fibrosis transmembrane conductance regulator (CFTR) to recipient cells in vitro. CFTR was purified from both CHO cells and Sf9 cells and reconstituted into two different types of vesicular delivery vehicles. In one, CFTR and HA were co-reconstituted into the same lipid vesicle. After binding to the cell surface, delivery of CFTR to the recipient cell was achieved by a transient, low-pH activation of the fusion activity of HA. A second delivery strategy used HA virosomes together with purified CFTR that had been reconstituted into vesicles containing gangliosides, a receptor for HA. After binding of the HA virosomes and CFTR-containing vesicles to the recipient cells, delivery to the plasma membrane again was achieved by a transient pH drop. Delivery of functional CFTR was assessed using the SPQ fluorescence assay. Functional CFTR was detected in a fraction (> 20%) of the recipient cells using this assay. Quantitative binding and fusion assays using radiolabeled virosomes and lipid vesicles showed that on the order of 1,000 of the added CFTR-containing vesicles bound to each C127 cell under the conditions of our delivery protocols. However, only a fraction of these vesicles fused and delivered CFTR to the cell plasma membrane. The two delivery strategies were found to be approximately equivalent in their ability to deliver active CFTR, and there were no significant differences between deliveries using purified CFTR from either cell source. These feasibility studies suggest that purified CFTR can be delivered to a recipient cell in a functional form and therefore represent a significant step in establishing the concept of protein replacement as a therapy for cystic fibrosis.

Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells.

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, alpha-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) Cell 68, 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-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-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.

Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro.

We have generated several clones of Chinese hamster ovary, mouse epitheloid C127, and pig kidney epithelial LLCPK1 cells producing high levels of functional recombinant human cystic fibrosis transmembrane conductance regulator (CFTR). Processing of CFTR to the mature and fully glycosylated form in these cells is inefficient with only approximately 40% of all newly synthesized CFTR being converted to the mature form. Furthermore, expression of the most frequent mutant allele of the cystic fibrosis (CF) gene, the delta F508 mutant in these epithelial cells, indicated that it is biosynthetically arrested at the endoplasmic reticulum and fails to traffic to the plasma membrane. Using a combination of CFTR mutants and monoclonal antibodies, all the detectable recombinant CFTR in these cells was determined at least under the conditions used, to be present as a monomer. To demonstrate the feasibility of protein replacement therapy, we were able to effect the physical transfer of functional recombinant CFTR produced in Chinese hamster ovary cells to the plasma membranes of Ha3b fibroblasts, a cell line devoid of cAMP-stimulated chloride channels. Transfer of CFTR was mediated by the hemagglutinin viral fusion protein of influenza virus present on the Ha3b cells. Efficiency of transfer was up to 25% of the target cells, and CFTR chloride channel activity was detectable for up to 12 h post-fusion. Therefore, with the development of an appropriate formulation of fusogenic proteoliposome or virosome containing reconstituted purified CFTR, it should be feasible to introduce functional CFTR into CF-affected cells.