Restoration of cyclic adenosine monophosphate-stimulated chloride channel activity in human cystic fibrosis tracheobronchial submucosal gland cells by adenovirus-mediated and cationic lipid-mediated gene transfer.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.

New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.

Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Characterization of an adenovirus gene transfer vector containing an E4 deletion.

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in the E4 region to contain only open reading frame 6. When assayed in cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, and virus yield are indistinguishable from wild type. Late protein synthesis is normal with the exception of fiber synthesis, which is reduced approximately 10-fold. Despite the reduced fiber synthesis, Ad2E4ORF6 viral particles appear to contain a full complement of fiber protein. Virus replication in cotton rats indicates that Ad2E4ORF6 is replication defective in vivo. This may have safety implications for the development adenovirus vectors in that virus arising by recombination in the E1 region of an Ad2E4ORF6-based vector would be defective for growth in vivo. The deletion of E4 open reading frames that are not required for virus growth in vitro increases the cloning capacity of adenovirus vectors by 1.9 kb and may be generally useful for the construction of adenovirus vectors containing large cDNA inserts and/or regulatory elements. We describe the inclusion of the A2E4ORF6 modification in a recombinant adenovirus vector, Ad2/CFTR-2, for gene transfer of the human cystic fibrosis transmembrane regulator (CFTR).

Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis.

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.

Cloning of the goat beta-casein-encoding gene and expression in transgenic mice.

The goat beta-casein-encoding gene (CSN2), which encodes the most abundant protein of goat milk, has been cloned and sequenced. The intron/exon organization of the 9.0-kb goat CSN2 gene is similar to that of other CSN2 genes. Expression of the goat gene was principally restricted to the mammary gland of lactating transgenic animals. A low level of expression was also observed in skeletal muscle and skin. In contrast to a rat CSN2 transgene [Lee et al., Nucleic Acids Res. 16 (1988) 1027-1041], the goat gene was expressed to a high degree in the lactating mammary gland. Differences in the content or context of regulatory elements may account for the enhanced performance of the goat relative to the rat CSN2 gene in transgenic mice.

Construction, expression and characterization of humanized antibodies directed against the human alpha/beta T cell receptor.

Completely humanized antibodies with specificity for the human alpha/beta TCR have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 CD regions and human EU framework regions were synthesized and replaced into previously isolated genomic fragments. These fragments were inserted into mammalian expression vectors containing the human kappa and gamma 1 C region exons. Two variants were constructed each containing selected BMA 031 amino acids within the human frameworks. The humanized genes were transfected into Sp2/0 hybridoma cells by electroporation and transfectomas secreting humanized antibody were isolated. Levels of antibody expression up to 7 pg/cell/24 h were obtained. The humanized antibody, BMA 031-EUCIV2, competed poorly with murine BMA 031 for binding to T cells. BMA 031-EUCIV3, however, bound specifically to T cells and competed effectively with both the murine BMA 031 antibody and a previously constructed chimeric BMA 031 antibody for binding to these cells. The relative affinity of BMA 031-EUCIV3 was about 2.5 times lower than BMA 031. The ability to promote antibody dependent cell-mediated cytolysis was significantly enhanced with the engineered antibodies as compared to murine BMA 031. Humanized BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft vs host disease, and autoimmunity.

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.

pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Bacteriophage P22 Cro protein: sequence, purification, and properties.

The DNA sequence of part of the bacteriophage P22 early regulatory region, including genes cro and c1, was determined. The protein product of the cro gene consists of 61 amino acid residues, and that of c1, 92 amino acid residues. Both genes were placed separately in plasmids from which they are expressed from a controllable promoter in vivo. Induced cells bearing the cro-expressing plasmid were used as a source for purifying and characterizing the Cro protein. The amino-terminal sequence of this protein was found to be as predicted by the DNA sequence; close agreement was also observed between its predicted and experimentally determined amino acid composition and molar extinction coefficient at 280 nm. In gel filtration experiments, Cro protein at concentrations around 10(-5) M appears to have a molecular weight of 8600, which is more consistent with monomers (6800) than with dimers (13 600). Cro protein binds specifically to the three repressor binding sites in the P22 right operator; in order of decreasing affinity, these are OR3, OR1, and OR2.

Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor.

The thermal denaturations of five revertant lambda repressors containing single amino acid substitutions in their N-terminal domains have been studied by differential scanning calorimetry. Two substitutions slightly decrease stability, and the remaining three render the protein more stable than wild type. The Gly48----Asn and Gly48----Ser proteins are 4 degrees C more stable than wild type. These two substitutions replace an alpha helical residue, and in each case a poor helix forming residue, glycine, is replaced by a residue with a higher helical propensity. We also present data showing that one revertant, Tyr22----Phe, has reduced operator DNA binding affinity despite its enhanced stability.

The lambda and P22 phage repressors.

The lambda cI repressor and the P22 c2 repressor contain two structural domains. In both proteins, the N-terminal domains mediate operator recognition and positive control of transcription, and the C-terminal domains mediate subunit oligomerization and recognition of the recA protein. In some cases, structural, biochemical, and genetic studies implicate particular repressor side chains in these processes.

Primary structure of the phage P22 repressor and its gene c2.

The amino acid sequence of the Salmonella phage P2 repressor and the DNA sequence of its gene c2 have been determined. Sequential Edman degradations on intact P22 repressor and repressor peptides generated by proteolytic and chemical cleavages have been overlapped to give approximately 97% of the complete protein sequence. Additionally, the nucleotide sequence of the P22 c2 repressor gene has been determined by DNA sequencing techniques. The DNA sequence and partial protein sequence are collinear and together define the complete amino acid sequence of P22 repressor. The repressor is a single-chain 216 amino acid polypeptide. Basic residues in the sequence tend to be clustered, and residues 9-20 are highly basic, containing five arginyl and three lysyl residues. The carboxyl-terminal 133 amino acids of the c2 repressor are homologous to the carboxyl-terminal sequence of the coliphage lambda cI repressor. The amino-terminal sequences of these two repressors show little similarity.