Huibi Cao

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation.

Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in bronchial epithelial cell lines. Treatment of these cells with IL-1β and TNF-α resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-κB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-κB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in Elf3 (homologous to human ESE-1) knockout mice, the expression of the inflammatory cytokine interleukin-6 (IL-6) is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with ∼6% of naive CD4+ T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4+ and CD8+ T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8+ T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.

Development of an inflammation-inducible gene expression system using helper-dependent adenoviral vectors.

Clinical studies have shown that gene therapy is a promising approach for treating such genetic diseases as the eye disease, Lebers congenital amaurosis. Development of gene therapy approaches for treating chronic inflammatory diseases is, however, more challenging because it requires the production of anti‐inflammatory molecules at the diseased tissues only when they are needed.

Knockdown of ZNF403 inhibits cell proliferation and induces G2/M arrest by modulating cell-cycle mediators

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

Subretinal gene delivery using helper-dependent adenoviral vectors

This study describes the successful delivery of helper-dependent adenoviral vectors to the mouse retina with long term and robust levels of reporter expression in the retina without apparent adverse effects. Since these vectors have a large cloning capacity, they have great potential to extend the success of gene therapy achieved using the adeno-associated viral vector.

Testing gene therapy vectors in human primary nasal epithelial cultures

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF.

Highly efficient retinal gene delivery with helper-dependent adenoviral vectors

There have been significant advancements in the field of retinal gene therapy in the past several years. In particular, therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses (AAV) to treat of a type of Lebers congenital amaurosis caused by RPE65 mutations. However, despite the success of retinal gene therapy with AAV, challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression. For example, Stargardts disease, a form of juvenile macular degeneration, is caused by defects in ABCA4, a gene that is too large to be packaged in AAV. Therefore, we investigated the ability of helper dependent adenovirus (HD-Ad) to deliver genes to the retina as it has a much larger transgene capacity. Using an EGFP reporter, our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1 × 105 infectious units and maintain transgene expression for at least 4 months. The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.

324. Successful Repeated Delivery of Helper-Dependent Adenoviral Vector Toachieve Efficient Long-Term Human CFTR Expression in Mouse Airwaythrough Transient Immunosuppression

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. As a chronic, lifelong disease, CF should be best treated with a continue level of CFTR expression. Pulmonary gene therapy may ultimately cure the CF lung disease. Thus, efficient long-term expression of the delivered CFTR gene in targeted cell types is essential to achieve this goal either by repeated application or with a long-duration expression system. Even though novel approaches to target airway progenitor/stem cells with gene editing may be more attractive, the efficiency for targeting specific progenitor cells will still be a challenge in airway delivery. Repeated administration of viral vectors may be required to increase the percentage of cells to be targeted and to boost long-term therapeutic effects. However, immune responses to viral proteins are the greatest barrier to repeated vector administration. Here, we demonstrated that repeated transduction of human CFTR gene in mouse airway can be successfully achieved with helper-dependent adenoviral (HD-Ad) vector through transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration and sustained expression through inhibition of host immune reactions. In this study, we examined cyclophosphamide effects on expression of the human CFTR in multiple rounds of HD-Ad vector delivery to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×1010 vector particles per mouse at day 0, day 70 and day 120. Two groups were treated with cyclophosphamide 6 hours before and 4 day and 8 days after vector delivery each time (except the last round of vector delivery for mice sacrificed at day 123). The other two groups without treatment were used as controls. One treated group and one control group were sacrificed at day 123, and the others at day 153 for detecting transgene expression and immune reaction. We found that cyclophosphamide has significantly improved the expression of CFTR (4.1 times higher) compared to the control group as determined by real-time qPCR 3 days after last delivery. And the CFTR expression level was 3.1 times higher 30 days after last vector transduction. Cyclophosphamide also significantly reduced the levels of antiadenoviral and neutralizing antibodies in both BALF and serum as well as prevented leukocyte infiltration in lung tissues. This data suggested that efficient repeated transduction of human CFTR gene with HD-Ad vector can be achieved by transient immunopuression in mouse lung.

574. Sustaining Helper-Dependent Adenoviral Vector-Mediated Human CFTR Expression in Mouse Airways Through Transient Immunosuppression

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. Gene therapy for CF lung disease has been tested extensively. However, sustained expression of the CFTR gene is a major challenge to gene delivery with either viral or no viral vectors. One strategy to improve the persistent CFTR expression is to modulate host immune responses by transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration, through inhibition of host immune reactions. In this study, we examined cyclophosphamide, dexmethoson, and combination of cyclosporine, methylprednsolone and azathioprine, for their effects on long term expression of the human CFTR delivered with helper-dependent adenoviral (HD-Ad) vectors to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×10E10 particles per mouse. Three groups were treated with immunesuppresants and one group without any treatment was used as a control. The immunosuppressants were administrated twice, at 6 hours before and 48 hours after nasal delivery of the HD-Ad-CFTR vector. Expression of the CFTR gene was assessed 4 months after vector administration. We found that cyclophosphamide has significantly improved the sustained expression of the CFTR gene (2.5 times higher) compared to the no treatment group as determined by real-time qPCR. It alsogreatly reduced the levels of anti-adenoviral and neutralizing antibodies in both BALF and serum. However, cyclophosphamide did not show major effects on the innate immune reaction to HD-Ad vectors, such as NK cell infiltration and activation, 4 days after vector delivery. Dexmethesone also greatly reduced neutralizing antibodies production, but not affected transgene expression. A combination of cyclosporine, methylprednsolone and azathioprine which is used to suppress immune reactions in lung transplantation, also did not affect CFTR gene expression, nor immune reactions following HD-Ad-CFTR vector delivery. These data demonstrate that transient administration of cyclophosphamide can significantly improve sustained HD-Ad-mediated transgene expression in mice and it is more effective than dexmethsone or the combination of cyclosporine, methylprednsolone and azathioprine.