Marlene S. Strayer

Perfluorochemical Liquid-Adenovirus Suspensions Enhance Gene Delivery to the Distal Lung

We compared lung delivery methods of recombinant adenovirus (rAd): (1) rAd suspended in saline, (2) rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2–4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.

SURFACTANT PROTEIN-B (SP-B) PROMOTER IN REPLICATION DEFICIENT ADENOVIRUS(rAd) DIRECTS PULMONARY EPITHELIAL CELL-SPECIFIC GENE EXPRESSION. • 1986

Gene therapy using rAd shows promise in treating genetic pulmonary diseases. Inherited deficiency of SP-B, a proteolipid critical to pulmonary surfactant, results in severe respiratory failure in term infants. SP-B is produced by alveolar type II cells and bronchiolar Clara cells and may be harmful within other cells due to its fusogenic properties. A SP-B promoter(SPB; -640/+319 relative to transcription start site) shows lung epithelial cell line specificity in transient transfection of H441 pulmonary adenocarcinoma cells with promoter strength similar to the promiscuous viral RSV promoter. To explore cell-type specific gene therapy for SP-B deficiency, we compared expression of the lacZ reporter gene driven by the RSV or SP-B promoters in rAd. Dose-response and time course experiments in H441 cells demonstrated efficient rAd.SPB.lacZ and rAd.RSV.lacZ infection with 24h of virus exposure at a multiplicity of infection (MOI) of≈1-10 with high-level β-galactosidase activity after 72h. X-gal staining showed specificity of rAd.SPB.lacZ for pulmonary derived cell lines (A549 and H441 versus HeLa) whereas all cell lines stained extensively with rAd.RSV.lacZ. This finding was confirmed by quantitative β-gal assay: the ratios of rAd.SPB.lacZ to rAd.RSV.lacZ β-gal activities(average ± SEM) were A549 = 1.96 ± 0.69; H441 = 0.66 ± 0.13; HeLa = 0.05 ± 0.01; p<0.05; n=3). Human fetal lung explants(24wk gestation) infected with rAd.SPB.lacZ had preferential X-gal staining of airway lining cells versus interstitial cells. Epithelia of explanted trachea showed minimal staining. All lung and trachea explant cell types stained extensively with rAd.RSV.lacZ. rAd.SPB.lacZ infection of mixed cells isolated from lung explants showed staining of most epithelial cells and rare fibroblasts. Primary fibroblasts infected with rAd.SPB.lacZ (MOI 1-300) had very low β-gal activities (7-94 A420/min/mg protein) whereas β-gal activities of fibroblasts infected with rAd.RSV.lacZ increased in a dose-dependent manner (480-55200 A420/min/mg protein). These results indicate that the SP-B promoter in rAd maintains cell line specificity and may be useful for limiting transgene expression to type II and Clara cells. (supported by 5 P30 HD28815)

Combinatorial Gene Therapy to Inhibit HIV: Improving Therapeutic Efficacy by Targeting Multiple Stages of the HIV-1 Replication Cycle Simultaneously

In these studies, we tested the effectiveness of combinatorial gene delivery to CCR5-expressing cell lines and primary cells to enhance resistance to HIV-1 infection. The transgenes used were chosen both to decrease membrane CCR5 and to inhibit HIV-1 replication, and were delivered using Tag-deleted SV40-derived vectors. rSV40s are very effective in transducing primary T cells and T cell lines. SV(RNAiR5), SV(RevM10.AU1) and SV(RNAiR5/RevM10.AU1) respectively deliver a small interfering RNA (siRNA) against CCR5, RevM10, to which a C-terminal AU1 epitope was added and which inhibits HIV-1 Rev, or both transgenes together. We used human cell lines (SupT1 and SupT1/CCR5) and primary human monocyte-derived macrophages (MDMs). We assessed RevM10 expression by flow cytometry (FACS). After transduction, we confirmed expression of each transgene by flow cytometry (FACS). Simultaneous delivery of both transgenes in a bifunctional vector protected both SupT1/CCR5 cells and MDMs from R5 tropic HIV-1Ba-L, better than either the monofunctional vectors, SV(RNAiR5) and SV(RevM10.AU1) individually. The bifunctional vector also protected from X4-tropic HIV-1, comparably to SV(RevM10.AU1). SV(RNAiR5) did not protect from X4-tropic HIV-1. Thus, combining these two transgenes in one vector protected from X4-tropic HIV-1 and provided enhanced protection for CCR5-bearing cells from R5-tropic strains of HIV-1. Combinatorial genetic therapy, by targeting of more then one HIV-1 function, may provide effective inhibition of HIV-1 replication.

589. Repeated Recombinant Adenovirus (rAd) Administration To Generate Mucosal Antibodies (Abs) vs Botulinum Toxin A (BoNT/A)

Many infectious agents enter the body via the mucosae. Thus, mucosal immunity is an important early defense and a logical vaccine target. Mucosal Abs (mainly secretory IgA, sIgA) are typically made after mucosal immunization. Adjuvants are often required to elicit a strong response. BoNT/A, one of the most potent poisons known, is a potential biological weapon. Since it can poison by inhalation, it could be aerosolized, easily disseminated and hard to inactivate.

Surfactant Protein B (SP-B) Promoter Function in Transgenic Mice |[bull]| 309

SP-B, a hydrophobic protein produced by alveolar type II and bronchiolar Clara cells, helps in the spreading and stability of surfactant film and is required for surfactant function. The SP-B gene is regulated both developmentally and hormonally. To study regulatory elements in the SP-B gene promoter in vivo, we made transgenic (tg) mice using a fragment of the human SP-B promoter (-1039/+431bp) linked to the chloramphenicol acetyl transferase (CAT) reporter gene. This SP-B DNA fragment contains both proximal and distal TTF-1 and HNF-3 transcription factor binding sites which promote lung epithelial cell line specificity in vivo. CAT activity in tg lung tissue was significantly increased compared to wild-type (22.2±3.2 vs 0.4±0.1 cpm/mg prot/min; p<0.01), whereas CAT activity in heart, liver, spleen, kidney, intestine, skeletal muscle and brain was not different. In two established tg lines CAT activity was found both in lungs (150 and 264 cpm/mg/min) and in trachea (50 and 125 cpm/mg/min), sites of type II and/or Clara cells in mice. Immunohistochemistry of lung sections using antibodies to CAT and human SP-B showed immunostaining for both proteins localized to type II and Clara cells. In tg fetal lungs, CAT activity was found at 17-19 days gestation and increased ≥4 fold (n=2) during explant culture. In tg adult lung explants (n=3), levels of CAT activity were maintained for at least 3 days (95±13% (se) of preculture). Transforming growth factor β(TGFβ1, 30 ng/ml), a cytokine which downregulates SP-B in human fetal lung explants, reduced CAT activity in both fetal (52±9% of control at 3 days, n=2) and adult mouse lung explants (60±11% of control at 3 days, n=1; 34±3% at 5 days, n=3) whereas35 S-methionine incorporation into total protein was unaffected(101±7%). In contrast, 12-O-tetradecanoyl phorbol-13 acetate (TPA, 30 nM), which also downregulates SP-B in human fetal lung explants, did not reduce CAT activity in tg adult lung ex vivo (182±54%, n=4). We conclude that the human SP-B promoter fragment (-1039/+431bp) exhibits tissue and lung cell specificity in vivo and confers developmental regulation and TGFβ responsiveness ex vivo. These transgenic animals provide a useful in vivo model to study specific regulatory elements in the SP-B promoter.