Andrea Pavirani

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.

cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.

Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression.

E1-deleted adenovirus (Ad) vectors expressing the human coagulation factor IX (hFIX) or the bacterial β-galactosidase (lacZ) were injected intravenously into various strains of immunocompetent (C57Bl/6, BALB/c, CD1, CBA/J, C3H) and immunodeficient (BALB/c-nu/nu, C57Bl/6-nu/nu, SCID, NIH-bg-nu-xid) mice. Regular analysis of mouse sera and tissues showed a persistent expression of both transgenes in immunodeficient mice, while detection diminished very rapidly in immunocompetent mice. The mechanisms responsible for the transient detection of the two transgenes were however not identical. Rapid decline of lacZ expression was correlated with a rapid decrease of viral DNA sequences, and consequently to the induction of a cellular immune response to the lacZ antigen. In contrast, absence of detectable levels of serum hFIX in immunocompetent animals was not associated with a loss of viral DNA but was strictly correlated with the induction of anti-hFIX antibodies. Surprisingly, anti-hFIX antibodies were never detected in C57Bl/6 mice, leading to prolonged detection of hFIX. These results suggest that cellular immunity to viral antigens plays a minor role in the early extinction of transgene expression and illustrate the influence of the cellular (eg lacZ) or humoral (eg hFIX) immunity to transgene-encoded products on the persistence of transgene expression.

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium.

The predicted protein domains coded by exons 9–12 and 19–23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide‐binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9‐ mRNA transcripts). In most (9 of 12), the exon 9‐ transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9‐ transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re‐evaluation of the same individuals 2–4 months later showed the same proportions of exon 9‐ transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon‐intron junctions relevant to exon 9 deletion (exon 8‐intron 8, intron 8‐exon 9, exon 9‐intron 9, and intron 9‐exon 10) were identical except for the intron 8‐exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9‐ transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype.

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.

High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland

The 5′ flanking region (6.3 kb) of the rabbit WAP (rWAP) gene possesses important regulatory elements. This region was linked to the human growth hormone (hGH) structural gene in order to target transgene expression to the mammary gland. Thirteen lines of transgenic mice were produced. Milk could be collected from six lines of transgenic mice. In five of them, hGH was present in the milk at high concentrations ranging from 4 to 22 mg ml−1. hGH produced by the mammary gland comigrated with hGH of human origin. It was biologically active, and through its prolactin-like activity induced lactogenesis when introduced into mammary culture media. Two of these mouse lines were studied further. hGH mRNA was only detected in the mammary gland during lactation. In the seven other transgenic lines, hGH was present in the blood of cyclic females. The prolactin-like effect of hGH in these mice probably induced female sterility, and milk could, therefore not be obtained. In two lines studied in more detail, the mammary gland was the main organ producing hGH, even in cyclic mice. Low ectopic expression was detected in other organs which varied from one line to the other. This was probably due to the influence on the transgene of the site of integration into the mouse genome. In the 13 lines studied, high mammary-specific hGH expression was not correlated to the transgene copy number. The rWAP-hGH construct thus did not behave as an independent unit of transcription. However, it can be concluded that the 6.3 kb flanking region of the rWAP gene contains regulatory elements responsible for the strong mammary-specific expression of hGH transgene, and that it is a good candidate to control high levels of foreign protein gene expression in the mammary gland of lactating transgenic animals.

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.

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5′ flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue‐specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co‐express the transgenes in hepatocytes were obtained. Hepatoma‐derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.