These studies were designed to test whether “gutless” rSV40 vectors carrying human CFTR cDNA could be effectively packaged and deliver CFTR expression after transduction, and whether the CFTR so provided constituted a functional chloride channel. It was important to ascertain that “gutless” rSV40 vectors carrying the 4.2 kb CFTR cDNA insert could be packaged by COS-7 cells. rSV40-CFTR was packaged by COS-7 cells at yields comparable to those obtained for other rSV40s that carry capsid genes: approximately 1011 IU/ml. Thus, the size of the vector genome, 5.5 kb, was within the packaging limits of this system. Some other vector systems require simultaneous cotransfection of packaging cells with multiple plasmids, or coinfection with helper viruses, in order to produce gutless vectors, but the effective packaging of a rSV40 lacking capsid genes in COS-7 cells indicates that the packaged rSV40 genomes need not carry SV40 capsid genes. The latter are expressed adequately, driven by their own promoter, by the COS-7 cells: neither helper virus nor cotransfection is involved. Preliminary studies show that SV40 capsid genes, under the control of the SV40 late promoter, are not expressed constitutively in COS-7 cells, but that the presence of a replicating rSV40 genome that includes the late promoter is sufficient to activate COS-7 transcription of the capsid genes in trans (M. Mariere and D.S. Strayer, unpublished data).
A possible explanation for this phenomenon is that replicating SV40 genomes titrate out a cellular repressor that inhibits transcription of integrated SV40 late genes (25
). In our system, then, the rSV40 genome replicating in COS-7 cells would bind the cellular repressor and, in so doing, trans-
activate the SV40 promoter to drive expression of the capsid genes. Replicating rSV40 genomes would then de-repress expression of SV40 capsid genes from the integrated copy of the wt SV40 genome in COS-7 cells. Thus, recombinant SV40-derived vectors do not need to carry SV40 capsid genes and gutless rSV40 vectors are made as efficiently as standard rSV40s.
CFPAC cells are a pancreatic adenocarcinoma line that carries the most common known loss of function mutation in the CFTR gene: ΔF508 (18
). The mutation causes an in-frame loss of a phenylalanine at position #508. The resulting abnormal protein can actually form a functional Cl- channel (26
), but loss of F508 causes the protein to be misfolded and consequently shunted to the cellular proteosome for degradation, so it does not reach the cell membrane to serve as an ion channel.
Transduction with rSV40-CFTR delivered detectable CFTR protein by immunostaining in vitro
and detectable transcription as determined by RT-PCR in vivo
. The in vitro
studies also demonstrated that rSV40 gene delivery transduced the great majority of cells in these cultures, without selection. This finding is consistent with previous data demonstrating that at rSV40s can deliver transgene expression efficiently and do not require the use of a selective agent (13
). It is useful to point out that expression of proteins delivered by rSV40s, while permanent, tends to be at lower levels than are seen using other gene delivery vector systems.
Most importantly, treatment with rSV40-CFTR provided a functional chloride channel activity that was lacking in both CFPAC and IB3 cells. Furthermore, channel function was demonstrated by two different assays of Cl- channel activity, both of which demonstrated similar functionality in the ion channel delivered by rSV40-CFTR. The level of Cl- channel activity in transduced cells was 1/3 to 1/2 of that seen in normal cells. Estimates of several investigators suggest that this level of Cl- channel activity is sufficient to avoid the most harmful consequences of mutation in CFTR (27
). Thus, if the level of activity seen here could be achieved and maintained in vivo,
it would be within the therapeutic range.
studies carried out in the bacterial agarose bead model lend further evidence of the functional and corrective nature of the rSV40 delivered CFTR gene. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted. Indeed, these studies, conducted by instillation of rSV40-CFTR into airways of CFTR-knockout mice, suggest that this vector may be able to provide significant functional correction in vivo
as well as in vitro
. Our studies report a statistically significant difference in weight loss between our two groups ~26% for the BUGT treated mice and ~19% for the CFTR rescued mice at day 3. In comparison, the original study using the pseudomonas bead model by Van-Heeckeren et al. reports a weight loss of ~18% in Cftr−/− mice versus ~13% in wild type mice (21
). In our experiments the weight loss was steeper and more prominent; this is probably due to the typical variability encountered in the preparations of the pseudomonas agar beads. However, one can appreciate the difference in weight loss between groups was proportional in the two separate studies. More importantly for CF related pathology rSV40-CFTR rescue resulted in the reduction of KC and IL-1B which correlated with the dramatic reduction of neutrophils influx to the lung and with a significantly improved overall inflammatory state of the lung as judged by histology.
CF gene therapy has stumbled at the transition between effective in vitro
and in vivo
gene delivery for many reasons (4
). Applying rSV40 vectors to the treatment of CF may help address some of these issues: impermanence of transgene expression (rSV40s integrate and express their transgenes indefinitely (12
), cytolytic inflammatory responses against transduced cells (cells transduced with rSV40s do not elicit cytotoxic responses (31
), and need for repeat dosing (rSV40s, alone of all viral gene delivery vectors, do not elicit neutralizing antibodies (16
). Other questions, such as the best route of administration (32
), e.g., the most appropriate target cell (33
) are dilemmas that have stimulated considerable thought and experimentation, but that remain to be resolved (10
Many hurdles remain before rSV40 gene delivery in CF can reach clinical fruition. Many issues, including vector safety and appropriate production procedures, all need to be considered. The unique ability of rSV40s to avoid eliciting neutralizing antibody has been demonstrated in rodents (12
), but needs to be confirmed in higher animals. Finally, the route of delivery remains a major concern, since neither aerosolization nor instillation of other vectors through the airways has not yet yielded promising results. Because SV40 is small (≈40 nm in diameter), it may be delivered more efficiently to the lungs in CF patients via the vascular system. We have administered rSV40 vectors carrying marker genes intravenously and detected abundant transgene expression in the pulmonary airway epithelium (34
Beyond questions of effectiveness, issues relating to vector safety are important. To date, our preparations of rSV40 vectors have been free of Tag+
revertant wtSV40s. Animal recipients of these vectors have not shown evidence of adverse effects or toxicity (13
Conventional therapies have improved survival for patients with CF dramatically. Interesting new approaches continue to offer benefits, or potential benefits, to some such individuals (35
). Some thoughtful approaches have recently been reported that may circumvent or decrease the barrier created by the airway inflammation and viscous mucous, so as to facilitate gene delivery (36
). However, gene therapy remains tantalizing, precisely because of its promise that CF treatment could one day actually give afflicted patients something approaching normal lives.
Whether rSV40 vectors make a contribution here remains to be seen. But our data suggest that they may provide an additional gene delivery option in treating cystic fibrosis.