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317. Analysis of rAAV Progenitor Cell Transduction in Mouse Lung
Preclinical Applications of AAV Vectors promoter for pancreas targeting. We also compared CMV and CB promoters for their transduction profiles. AAV7, AAV8 or AAV9 vectors containing different expression cassettes were administered intravenously to adult C57BL6 mice at different doses. nLacZ expression and vector genome persistence in different tissues were evaluated morphologically and quantitatively at day 28. Our study identified a set of expression cassettes that can accomplish efficient tissue-specific expressional targeting to different tissues of adult mice by AAV-mediated gene transfer, which has important implications in creation of animal models for tissue-specific somatic transgenics or knock down. Unique transgene expression profiles of CMV and CB expression cassettes in mouse tissues as well as correlation between tissue-specific expression and transgene-specific T cell response were also investigated.
317. Analysis of rAAV Progenitor Cell Transduction in Mouse Lung
Xaioming Liu,1,2 Meihui Luo,1 Ziying Yan,1,2 Chenhong Guo,1,3 Yujiong Wang,3 John F. Engelhardt.1,2 1 Department of Anatomy & Cell Biology, The University of Iowa, Iowa City, IA; 2Center for Gene Therapy, The University of Iowa, Iowa City, IA; 3College of Life Science, Ninxia University, Yinchuan, Ninxia, China. To investigate whether rAAV vectors transduce progenitor/stem cells in the lung, we evaluated the life span and phenotype of rAAV transduced cells in the lung using a LacZ-CRE reporter transgenic mouse model and Cre expressing rAAV. In this model, expression of CRE recombinase led to LacZ genetic marking of transduced cells and their descendants, allowing for lineage tracking of potential progenitor/stem cells. 6-10 week old adult floxed/stop-LacZ-CRE reporter mice were infected via the trachea with 2 x 1010 particles of rAAVCre virus pseudotyped in AAV1, 2, or 5 capsid. The relative β-galactosidase (LacZ) activity in the lung was examined by measuring enzymatic activity and X-gal staining at 1 to 24 weeks postinfection. Our study demonstrated that both rAAV serotypes 1 and 5 vector were able to efficiently express the Cre transgene in alveolar cells, which resulted in long-term stable and increasing LacZ activity in the lung to 6 months (at which time the experiment was terminated). rAAV5Cre infection gave rise to more rapidly and efficient recovery of LacZ activity than did the rAAV1Cre vector. LacZ activity recovered by transduction with rAAV1Cre and rAAV5Cre was 3% and 5% as compared to that of the Rosa26-LacZ functional reporter mouse lung, respectively. In contrast, rAAV2CRE demonstrated little LacZ activity above that of mock-infected mice, with only a few of X-gal staining positive cells in the conducting and alveolar airway epithelia. To investigate whether airway progenitors were transduced with rAAV, we injured the airways of rAAV infected mice with Naphthalene at 2 weeks post-infection while simultaneously labeling with 5-bromodeoxyuridine (BrdU) to capture slow cycling progenitor/stem cells that entered the cell cycle and retain label. Characterization of Cre-rescued LacZ positive cells was performed by co-localizing surfactant protein C (SP-C) and BrdU at 60-90 days post BrdU labeling. In this context, a subset of LacZ positive cells were also SP-C positive and retained BrdU label. These data suggest that both rAAV1 and rAAV5 may be capable of transducing long-lived, slowly replicating, alveolar type II progenitor/stem cell in the lung. In contrast to the distal lung, transduction of the conducting proximal airways was much less efficient for both rAAV1 and 5, given rise to abundant LacZ positive epithelial cells only at the high dose of 2 x 1011 particles for rAAV1Cre. Furthermore, rescued LacZ positive cells were much less persistent in the proximal airways and BrdU positive label-retaining cells were LacZ negative, indicating that only differentiated and potentially short-lived transient amplifying cells were efficiently infected in the conducting airways with both serotypes. This alternative approach to evaluating rAAV transduction Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
may be useful in determining the half-life of cells infected with rAAV and whether these cell types are progenitor/stem cells. Additionally, this approach may help dissect immunologic mechanisms that lead to clearance of viral infected cells and/or transgene expressing genomes.
318. AAV5 Intraarticular Administration of TNF Small Interfering RNA Prevents Progression of Arthritis
Maroun Khoury,1 Gabriel Courties,1 Margriet J. B. M. Vervoordeldonk,2 Paul P. Tak,2 Christian Jorgensen,1 Florence Apparailly.1 1 U844, Inserm, UM1, CHU Lapeyronie, Montpellier, France; 2 Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Arthrogen BV, Amsterdam, The Netherlands.
RNA interference (RNAi) has rapidly become a powerful tool for drug target discovery, and interest is rapidly growing in extension of its application to animal disease models. To overcome the limitations of in vivo RNAi delivery, several viral vectors are used for their efficacy to deliver short hairpin small interfering (si)RNAs (shRNAs), resulting in long term silencing. The successful advanced strategies in rheumatoid arthritis (RA) gene therapy using safe viral vectors such as adeno-associated viruses (AAVs) prompted us to determine the therapeutic potential of a recombinant AAV type 5 (rAAV5) expressing shRNAs for TNF-α (rAAV5-shTNF), a key pro-inflammatory cytokine in RA. First, we have cloned two different shRNA sequences silencing the TNF (named TNF1 and TNF2) at the mRNA level into a rAAV5-GFP vector, under the control of a H1 promoter. The corresponding plasmids have been validated in vitro for the dose-dependent silencing of the targeted mRNA by real-time PCR following transient transfection of the LPS-challenged mouse macrophage cell line J774. When collagen-induced arthritic mice received a single intraarticular administration of both rAAV5shTNF vectors (5 x 109 ip/joint), disease incidence and severity were dramatically decreased in the injected joints compared with an empty rAAV5-H1-GFP used as control. The disease incidence in the rAAV5-shTNF1/2-treated group reached 43% compared with the control groups where 100% of mice developed clinical signs of arthritis by the end of experiment (p<0.001). Moreover, rAAV5shTNF1/2 stabilized paw swellings from day 28 until sacrifice, while swelling increased in both control groups. Interestingly, the protective effect was restricted to the injected joints while uninjected ones showed arthritic scores comparable to controls. The therapeutic effect was associated with local down-regulation of both inflammatory and autoimmune components of the disease, including inhibition of antigen-specific T-cell proliferation and regulation of Th1/Th2 balance, while the systemic T-cell proliferation and pro-inflammatory cytokines were not affected. Our data present the first proof-ofprinciple for the application of an AAV5-siRNA-based therapy as a local TNF blockade in RA.
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317. Analysis of rAAV Progenitor Cell Transduction in Mouse Lung
Xaioming Liu,1,2 Meihui Luo,1 Ziying Yan,1,2 Chenhong Guo,1,3 Yujiong Wang,3 John F. Engelhardt.1,2 1 Department of Anatomy & Cell Biology, The University of Iowa, Iowa City, IA; 2Center for Gene Therapy, The University of Iowa, Iowa City, IA; 3College of Life Science, Ninxia University, Yinchuan, Ninxia, China. To investigate whether rAAV vectors transduce progenitor/stem cells in the lung, we evaluated the life span and phenotype of rAAV transduced cells in the lung using a LacZ-CRE reporter transgenic mouse model and Cre expressing rAAV. In this model, expression of CRE recombinase led to LacZ genetic marking of transduced cells and their descendants, allowing for lineage tracking of potential progenitor/stem cells. 6-10 week old adult floxed/stop-LacZ-CRE reporter mice were infected via the trachea with 2 x 1010 particles of rAAVCre virus pseudotyped in AAV1, 2, or 5 capsid. The relative β-galactosidase (LacZ) activity in the lung was examined by measuring enzymatic activity and X-gal staining at 1 to 24 weeks postinfection. Our study demonstrated that both rAAV serotypes 1 and 5 vector were able to efficiently express the Cre transgene in alveolar cells, which resulted in long-term stable and increasing LacZ activity in the lung to 6 months (at which time the experiment was terminated). rAAV5Cre infection gave rise to more rapidly and efficient recovery of LacZ activity than did the rAAV1Cre vector. LacZ activity recovered by transduction with rAAV1Cre and rAAV5Cre was 3% and 5% as compared to that of the Rosa26-LacZ functional reporter mouse lung, respectively. In contrast, rAAV2CRE demonstrated little LacZ activity above that of mock-infected mice, with only a few of X-gal staining positive cells in the conducting and alveolar airway epithelia. To investigate whether airway progenitors were transduced with rAAV, we injured the airways of rAAV infected mice with Naphthalene at 2 weeks post-infection while simultaneously labeling with 5-bromodeoxyuridine (BrdU) to capture slow cycling progenitor/stem cells that entered the cell cycle and retain label. Characterization of Cre-rescued LacZ positive cells was performed by co-localizing surfactant protein C (SP-C) and BrdU at 60-90 days post BrdU labeling. In this context, a subset of LacZ positive cells were also SP-C positive and retained BrdU label. These data suggest that both rAAV1 and rAAV5 may be capable of transducing long-lived, slowly replicating, alveolar type II progenitor/stem cell in the lung. In contrast to the distal lung, transduction of the conducting proximal airways was much less efficient for both rAAV1 and 5, given rise to abundant LacZ positive epithelial cells only at the high dose of 2 x 1011 particles for rAAV1Cre. Furthermore, rescued LacZ positive cells were much less persistent in the proximal airways and BrdU positive label-retaining cells were LacZ negative, indicating that only differentiated and potentially short-lived transient amplifying cells were efficiently infected in the conducting airways with both serotypes. This alternative approach to evaluating rAAV transduction Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
may be useful in determining the half-life of cells infected with rAAV and whether these cell types are progenitor/stem cells. Additionally, this approach may help dissect immunologic mechanisms that lead to clearance of viral infected cells and/or transgene expressing genomes.
318. AAV5 Intraarticular Administration of TNF Small Interfering RNA Prevents Progression of Arthritis
Maroun Khoury,1 Gabriel Courties,1 Margriet J. B. M. Vervoordeldonk,2 Paul P. Tak,2 Christian Jorgensen,1 Florence Apparailly.1 1 U844, Inserm, UM1, CHU Lapeyronie, Montpellier, France; 2 Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Arthrogen BV, Amsterdam, The Netherlands.
RNA interference (RNAi) has rapidly become a powerful tool for drug target discovery, and interest is rapidly growing in extension of its application to animal disease models. To overcome the limitations of in vivo RNAi delivery, several viral vectors are used for their efficacy to deliver short hairpin small interfering (si)RNAs (shRNAs), resulting in long term silencing. The successful advanced strategies in rheumatoid arthritis (RA) gene therapy using safe viral vectors such as adeno-associated viruses (AAVs) prompted us to determine the therapeutic potential of a recombinant AAV type 5 (rAAV5) expressing shRNAs for TNF-α (rAAV5-shTNF), a key pro-inflammatory cytokine in RA. First, we have cloned two different shRNA sequences silencing the TNF (named TNF1 and TNF2) at the mRNA level into a rAAV5-GFP vector, under the control of a H1 promoter. The corresponding plasmids have been validated in vitro for the dose-dependent silencing of the targeted mRNA by real-time PCR following transient transfection of the LPS-challenged mouse macrophage cell line J774. When collagen-induced arthritic mice received a single intraarticular administration of both rAAV5shTNF vectors (5 x 109 ip/joint), disease incidence and severity were dramatically decreased in the injected joints compared with an empty rAAV5-H1-GFP used as control. The disease incidence in the rAAV5-shTNF1/2-treated group reached 43% compared with the control groups where 100% of mice developed clinical signs of arthritis by the end of experiment (p<0.001). Moreover, rAAV5shTNF1/2 stabilized paw swellings from day 28 until sacrifice, while swelling increased in both control groups. Interestingly, the protective effect was restricted to the injected joints while uninjected ones showed arthritic scores comparable to controls. The therapeutic effect was associated with local down-regulation of both inflammatory and autoimmune components of the disease, including inhibition of antigen-specific T-cell proliferation and regulation of Th1/Th2 balance, while the systemic T-cell proliferation and pro-inflammatory cytokines were not affected. Our data present the first proof-ofprinciple for the application of an AAV5-siRNA-based therapy as a local TNF blockade in RA.
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