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Adeno-associated virus type 2 (AAV) vectors have been used for gene expression in respiratory epithelial cells and may be useful in gene therapy for diseases like cystic fibrosis (CF) which affect the airways. The AAV p5 promoter together with the AAV inverted terminal repeat (ITR) forms a 263-base pair cassette which mediated efficient expression in a CF bronchial epithelial cell line. We report here that the ITR itself can mediate gene expression. In stable transfection assays, AAV-CF vectors expressing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA from either the p5 promoter or the ITR restored cAMP regulation of the chloride efflux characteristic of CFTR function. An AAV-ITR-CF vector deleted for the amino terminus of CFTR was also functional. This vector was packaged into AAV particles and used to transduce cells without selection. Transduced cells also exhibited cAMP-regulated Cl- efflux. The complemented cell lines showed increased levels of CFTR protein immunofluorescence, and the presence of intact AAV-CF vector sequence was confirmed by Southern blot analysis of rescued vector sequences. These studies provide novel insights into AAV gene expression, and this newly described promoter allows for the production of AAV vectors expressing CFTR in those differentiated cells affected in CF.

The past decade of research in cystic fibrosis has produced a wealth of information about the underlying defect responsible for the disease. The initial finding that the physiological disturbance in CF is one of abnormal electrolyte transport across epithelial tissues led to the elucidation of a pathway in which epithelial chloride transport is normally elicited in response to beta-adrenergic stimuli and involves the second messenger cAMP to activate protein kinase A. While that pathway was being described, work on the genetic front was concurrently providing information about the genomic location of the gene causing CF, which ultimately led to the identification and cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator. The cloned CFTR gene provided a powerful reagent to use in the next generation of cell physiology experiments, in which it was determined that CFTR is not only the substrate of PKA phosphorylation, a step previously determined to be in the activation pathway of the chloride channel, but is in fact a cAMP-dependent chloride conducting channel itself. Further analysis of the gene has shown that although there is a single mutation that accounts for most of CF, there are well over 200 other lesions within the gene that can cause disease as well. Identification of these mutations has provided information into the normal function of CFTR by studying these variants in heterologous expression systems. As a result, the molecular mechanism of CFTR function is beginning to unfold, as well as the mechanism by which particular mutations impair that function. From a clinical perspective, the research brings optimism from two directions. First, understanding how disease-causing mutations impair function may culminate in pharmacologic approaches that can restore function to some of these mutants. Second, treating the disease at the level of the gene appears to be a realistic goal: Gene transfer experiments in cultured CF cells have shown that the procedure will restore cAMP-dependent chloride conductance to the cells, laying the groundwork for somatic cell gene therapy as a feasible treatment for CF. Currently, work is rapidly progressing in developing delivery systems for this purpose. Finally, animal models that should not only aid in understanding the physiology of electrolyte transport in epithelia but should serve as indicators for tests of therapeutic approaches to treating CF are being developed, either by pharmacological means or by gene delivery protocols.(ABSTRACT TRUNCATED AT 400 WORDS)

We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

Serum amyloid A (SAA) is a major acute-phase plasma protein synthesized by the liver. In addition to the two major plasma isoforms described in humans (SAA1 and SAA2), a third form (SAA3) has been demonstrated in several other species and is distinguished by predominant extrahepatic expression. Two clones, Ch11g5-1-1 and HDg1-1, containing the human SAA3 gene are described in this report. The human SAA3 gene is comparable in organization to the SAA1 and SAA2 genes and shares with them 87% nucleotide identity in the region spanning exon 3 through exon 4. Sequences 5' to exon 3, however, are strikingly different from those in the SAA1 and SAA2 genes. For instance, the sequence deduced for amino acids 1-12 (exon 2) has only 25% identity with human SAA1 and SAA2; it most closely resembles that of rabbit SAA3 isolated from synovial fibroblast cultures (75% identity). Although rabbit SAA3 induces collagenase production in an autocrine fashion the human SAA3 gene is not expressed. This is shown by: (i) a single base insertion in the sequence corresponding to codon 31, (ii) the inability of a 918-bp fragment immediately upstream from SAA3 exon sequences to direct transcription of a chloramphenicol acetyltransferase reporter gene, and (iii) the absence of detectable human SAA3 in mRNA.

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.

We have used retrovirus-mediated gene transfer to demonstrate complementation of the cystic fibrosis (CF) defect in vitro. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC-1, a pancreatic adenocarcinoma cell line derived from a patient with CF that stably expresses the chloride transport abnormalities characteristic of CF. CFPAC-1 cells were exposed to control virus (PLJ) and CFTR-expressing virus (PLJ-CFTR); viral-transduced clones were isolated and subjected to molecular and physiologic analysis. RNA analysis detected a viral-derived CFTR transcript in all of the PLJ-CFTR clones that contained unrearranged proviral sequences. Agents that increase intracellular cAMP stimulated 125I efflux in PLJ-CFTR clones but not PLJ clones. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses were due to cAMP stimulation of Cl conductance. Our findings indicate that expression of the normal CFTR gene confers cAMP-dependent Cl channel regulation on CF epithelial cells.

Using chromosome jumping and walking and restriction fragment length polymorphism (RFLP) analysis, we have defined the region which must contain the cystic fibrosis gene. DNA segments spanning approximately 250 kb in the direction of the gene were isolated and used to identify several new polymorphisms informative in cystic fibrosis families. These RFLPs include a highly polymorphic, CA/GT repeat, and a 10 bp insertion uncovered using the polymerase chain reaction. By analyzing a family with a recombination near the gene, we can exclude this region as containing the mutation. Data on the extent of linkage disequilibrium of these markers provides additional information on where the gene is located.

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as "reverse genetics," in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of its usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should now be applicable to any genetic locus for which a closely linked DNA marker is available.

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.

The gene for Huntington disease (HD) has been localized to the distal portion of the short arm of human chromosome 4 by linkage analysis. Currently, the two closest DNA markers are D4S10 (G8), located approximately equal to 3 centimorgans centromeric to HD, and D4S43 (C4H), positioned 0-1.5 centimorgans from HD. In an effort to move closer to the HD gene, with the eventual goal of identifying the gene itself, we have applied the technique of chromosome jumping to this region. A 200-kilobase jumping library has been constructed, and a jump from D4S10 has been obtained and its approximate distance verified by pulsed field gel electrophoresis. Two restriction fragment length polymorphisms have been identified at the jump locus, which is denoted D4S81. Linkage analysis of previously identified recombinants between D4S10 and HD or D4S10 and D4S43 shows that in two of five events the jump has crossed the recombination points. This unequivocally orients D4S10 and D4S81 on the chromosome, provides additional markers for HD, and suggests that recombination frequency in this region of chromosome 4 may be increased, so that the physical distance from D4S10 to HD may not be as large as originally suspected.

The gene for cystic fibrosis (CF) is known to be flanked by the closely linked DNA markers met and J3.11 on chromosome 7. Using the technique of pulsed-field gel electrophoresis, we have constructed a complete overlapping restriction map of approximately 3000 kb of DNA in this region. The met and J3.11 probes are found to be between 1300 and 1800 kb apart, which compares well with their genetic distance of 1-2 cM. The CF gene must be located within this interval, and the availability of this physical map should be of considerable utility in mapping additional clones as the search for the gene proceeds.

The cystic fibrosis (CF) locus has been located, by both linkage analysis and physical mapping, to a 900-kb region of 7q22-31 flanked by D7S8 (J3.11) and D7S23 (XV-2c). Using a 100-kb general jumping library, we isolated two sequential jump clones, J31 and J29, to one side of the D7S8 region and one jump clone, J32, to the other side of D7S8, so that the total region covered is about 300 kb. Three new RFLPs were detected by J29 and J32. Using PFGE mapping and the three jump clones, we found it possible to orient D7S8 on the chromosome and, by linkage analysis, to further narrow the CF region by 100 kb. The orientation of D7S8 will be useful for directing the isolation of other jump clones toward the CF locus. Though the newly described RFLPs are in considerable linkage disequilibrium with D7S8 polymorphisms, they increase the informativeness of genetic markers in the D7S8 region and should be useful in prenatal diagnosis.