72 Transgene Expression in Murine Respiratory and Digestive Organs Following In Utero Delivery of Lentiviral Vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Societ[.]
LUNG AND RESPIRATORY DISEASE 70 Restoration of Ciliary Activity to Primary Ciliary Dyskinesia Cells Using an EIAV-Based Lentivirus Vector Lawrence E Ostrowski,1 John C Olsen.1 Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC Primary Ciliary Dyskinesia (PCD) is caused by mutations in genes that impair the function of motile cilia The disease is inherited in an autosomal recessive pattern and is heterogeneous at both the genetic and the phenotypic level Patients with PCD suffer from recurrent otitis media, chronic rhinosinusitis, and chronic respiratory infections, leading to bronchiectasis At present, there is no cure and no specific treatments for PCD We have developed a murine model for PCD by introducing flanking loxP targeting sequences around two exons of the dynein intermediate chain, Dnaic1 This gene encodes a protein that is part of the outer dynein arm assembly in respiratory cilia, and is mutated in 10-20% of human cases of PCD Deletion of Dnaic1 by tamoxifen activation of Cre recombinase (CreER) results in essentially complete ciliary immotility in differentiated cultures of murine tracheal epithelial cells In this work, we have used an influenza HA-pseudotyped EIAV-based lentivirus vector to transfer a normal copy of the murine Dnaic1 gene to PCD cells and restore ciliary activity Mouse tracheal epithelial cells from Dnaic1/floxed mice expressing CreER were cultured at an air/liquid interface on collagen-coated porous membranes Cultures were treated with tamoxifen to induce the deletion of Dnaic1 and generate PCD cultures Cultures were transduced from the apical surface with vector encoding the Dnaic1 gene expressed from the human ciliated cell-specific FOXJ1 promoter or the ubiquitously expressed chicken beta-actin promoter Control cultures were untreated or were treated with tamoxifen and a control vector expressing only EGFP Ciliary activity was quantified using high-speed video microscopy Cultures treated with the vectors encoding the normal Dnaic1 gene demonstrated a significant increase in ciliary activity (up to 100-fold) compared to control cultures treated with vector only expressing EGFP The ciliary beat frequency of the corrected cells was not significantly different from that of control cultures not treated with tamoxifen (10.2 +/- 0.6 Hz compared to 11.8 +/- 0.5 Hz) These results show that a normal ciliary protein expressed from a lentivirus vector can be incorporated correctly into the complex ciliary axoneme and restore function These studies suggest that gene transfer may be a viable treatment option for patients with PCD 71 High Efficiency Gene Transfer to Airways of Mice Using Influenza Hemagglutinin Pseudotyped EIAV-Based Lentivirus Vectors Manij Patel,1 Angela M Giddings,1 John C Olsen.1 Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC One limitation to airway gene transfer has been attributed to the lack of receptors on the luminal surface of polarized epithelial cells to commonly used viral envelopes A potential way of overcoming this limitation is by pseudotyping lentiviral vectors with the influenza hemagglutinin (HA) glycoprotein Improvements in production of HA-pseudotyped equine infectious anemia virus (EIAV)-based lentivirus vectors yield concentrated vector preparations having titers in excess of 109 IU/ml Administration of HA-pseudotyped luciferase or lacZ vectors to mice by nasal inhalation results in high level expression in the nose, trachea, and lungs From X-Gal stained histological lung sections, 60-70% of small or large airways showed at least some evidence of lacZ expression Histological sections from 7-week old CD-1 mice dosed at weeks of age showed increased cellularity near regions of high lacZ gene expression suggesting a mild inflammatory response Despite this evidence of inflammation, vector S30 transgene expression persisted for over months although the levels of gene expression decreased steadily over time We hypothesized that this decrease in expression was related to the inflammation we observed and postulated that the decline in gene expression was due to activation of a cell-mediated immune response against cells expressing the transgene As previous work involving gene transfer to the liver has suggested that inadvertent gene transfer to cells of hematopoietic lineage at the time of gene transfer could lead to more robust immune clearance of transduced cells, we tested whether suppressing expression in hematopoietic cells might increase the persistence of airway expression To this, we used the strategy of Brown and colleagues to specifically suppress expression in hematopoietic lineages (Brown, BD, Venneri, MA, Zingale, A, Sergi Sergi, L, and Naldini, L (2006) Nat Med 12 585-91) Four tandem copies of a 23-nucleotide target sequence (T142-3) of a naturally occurring miRNA (mir 142-3p) that is expressed in hematopoietic cells were introduced into an EIAV vector The T142-3 sequence was placed 3’ of a woodchuck hepatitis virus post-transcriptional regulatory element in a vector that expresses luciferase from a hybrid CMV/beta-actin promoter In vitro comparison of vectors with and without T142-3 in human monocytic U937 cells that express miR1423 indicated a 20-fold knock down of luciferase activity from the vector containing the T142-3 sequence In contrast, no significant differences in expression were observed in non-hematopoietic human cells A further comparison of vector expression was pursued in vivo in the nasal epithelia of CD-1 mice Luciferase expression levels were similar in both sets of mice shortly after transduction By nine months, expression from vectors that contained the T142-3 sequence had decreased 10-fold from original levels whereas vectors without the T142-3 sequence had decreased 200-fold This significant difference in expression levels is consistent with the notion that incorporation of T142-3 sequences into lentiviral vectors decreases activation of the immune response in airway gene transfer 72 Transgene Expression in Murine Respiratory and Digestive Organs Following In Utero Delivery of Lentiviral Vectors Suparna Mishra,1 Xingchao Wang,1 Nancy Smiley,1 Dinithi Senadheera,1 Carolyn Lutzko.1 Saban Research Institute, Childrens Hospital of Los Angeles, Los Angeles, CA Cystic Fibrosis (CF) is the most common genetic disorder in the U.S affecting 1/2500 individuals per year CF is caused by mutations in the CFTR gene encoding a chloride ion channel present in epithelial cells lining various organs such as the lungs, intestine, pancreas and liver Although mortality in CF patients is primarily a result of lung failure, significant morbidity results from malfunction of the nonrespiratory organs To find a comprehensive treatment for CF that will prevent disease symptoms and pathology in all affected organs, we have delivered lentiviruses carrying test genes in utero at E12.5, E14.5 and E16.5 We then evaluated the efficiency of transgene expression in all CF affected organs The results of our studies demonstrated luciferase gene expression up to one month following in utero delivery at E16.5 Whole body images showed bioluminescent signal in the thoracic and abdominal cavities and organ imaging identified these organs as lung, stomach and intestine To further identify the cell types expressing the transgene, EGFP containing lentiviral vectors were delivered at E14.5 and E16.5 Double immunofluorescence was used to characterize the EGFP expressing cells in the lung, trachea, intestine, liver and skin Half of the mice (6/13) had an average of 6% of their lung and tracheal epithelial cells expressing EGFP; a third (4/13) of the mice expressed EGFP in non-epithelial cells of the skin and liver and one mouse showed EGFP expression in the epithelial cells of the intestine EGFP expression was absent in the pancreases of these mice Quantitative PCR demonstrated that the average proviral Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy RNA VIRUS VECTORS I copy/cell in the various organs ranged from 1/1 to 1/100,000 There was no difference in EGFP biodistribution and expression pattern between the E14.5 and E16.5 lentiviral deliveries, although high mortality was associated with E12.5 delivery Together, our results demonstrate efficient gene transfer to and expression in the respiratory epithelium in the predicted therapeutic range RNA Virus Vectors I 73 TTRAP Enhance the Lentiviral Gene Expression Xu Wang, Jinzhong Chen, Jinglun Xue Institute of Genetics, Fudan University, Shanghai, China The lentivirus integrase HIV-IN plays a crucial role in the viral life cycle, especially the step of integration However, the cellular response to the incoming viral protein and potential interaction between IN and cellular proteins is largely unknown This study reveals that HIV integrase interacts with a recently identified PML-NBs associated protein TTRAP by yeast two hybrid, coimmunoprecipitation and intracellular co-location Knocking down the expression of endogenous TTRAP with specific siRNA duplex decreased the lentiviral integration rate, while TTRAP over expression increased the integration apparently Interestingly, TTRAP facilitates the lentiviral vector integration in a PML–independent manner Knocking down the expression of PML presents no difference in integration comparing with control group This is the first time that HIV integrase is reported to interact with a PML-NBs protein which facilitates the lentivirus integration The result provided a clue to improve the lentiviral vector integration or to develop the therapy methods against HIV infection 74 A Daxx-Binding Tetrapeptide 870KELK873 of HIV-1 Integrase Is Critical for Lentiviral Gene Expression Limin Wan, Jinzhong Chen Insititute of Genetics, Fudan University, Shanghai, China Our recent studies have found cellular protein Daxx to interact with HIV-1 integrase, but the region critical for this interaction has not been identified Here we found that a tetrapeptide 870KELK873 in HIV-1 integrase C-terminus is crucial for Daxx-binding Further investigation on the functions of this region suggested that deleting the tetrapeptide affects neither virus packaging nor viral gene integration into the host genome However, the reporter gene EGFP expression was significantly lower in cells infected by the HIV-1derived lentivirus without the tetrapeptide compared to those infected by wild-type lentivirus Our results suggest that the Daxx-binding tetrapeptide 870KELK873 of HIV-1 integrase is critical for the HIV1-derived lentiviral reporter gene expression, which may shed some light on the gene therapy based on lentiviral vectors 75 Sp100 Inhibited the Lentiviral Gene Expression Liping Qu, Jinzhong Chen Institute of Genetics, Fudan University, Shanghai, China The lentivirus integrase HIV-IN plays a crucial role in the viral life cycle, especially the step of integration But the cellular response to the incoming viral protein and potential interaction between IN and cellular proteins is unknown This study reveals that HIV integrase interacts with a PML-NBs associated protein Sp100 by yeast two hybrid, co-immunoprecipitation and intracellular co-location Knocking down the expression of endogenous Sp100 with specific siRNA duplex increased the lentiviral gene expression, while Sp100 over expression decreased the lentiviral gene expression apparently Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy The result provided a clue to improve the lentiviral vector integration or to develop the therapy methods against HIV infection 76 Enhancing Retroviral Transduction of Cord Blood CD34+ Cells by MIP-1α Leili Moezzi,1 Kamran Alimoghaddam,2 Seyed Hamidolah Ghaffari,2 Alireza Ardjmand,2 Pantea Ghodsi,2 Bahram Chahardouli,2 Somayeh Shahrokhi,3 Ardeshir Ghavamzadeh.2 Tehran University of Medical Sciences, Faculty of Paramedical Sciences, Tehran, Islamic Republic of Iran; 2HematologyOncology and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; 3Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran Introduction: Hematopoietic stem cells are in quiescence state and because of requirement of retroviral transduction to infect dividing cells; they are resistant to retrovirus transduction and needs to pre-stimulation by a cytokine cocktail For proliferation without maturation, we suggest MIP-1α as a novel factor Material and methods: retroviral vector produced by PG13/LN C8 cells tittered on hela cells Then CD34+ cells of cord blood prestimulated in serum free media supplemented with 50 ng/ml SCF,Flt3,TPO,IL6 in the presence and absence of 50 ng/ml MIP-1plus 8g/ml protamin sulfate and viral supernatant media) with and reefed them after 24 and 72 hours Then cells were harvested from the plate and seeded into new dishes in the absence of viral supernatant for one week Transduction efficiency by semi sensitive PCR for Neomycin resistance gene was assessed Results: Multiplicity of infection was 1.7105 PCR analysis of neomycin resistance gene in cord blood CD34+ cells in the presence and absence of MIP-1revealed improved Transduction of cord blood cells (CD34+ = 40.7%, CD34+MIP=65%) Conclusion: Addition of MIP-1α to cytokine cocktail may improve transduction efficiency of cord blood hematopoietic progenitor cells Further studies required for clarify its effect on functional properties of CD34+ cells 77 CCR5 Positive Cell Specific Targeted Transduction of a CCR5 shRNA Lentiviral Vector for HIV Gene Therapy Joseph S Anderson,1 Jan Nolta,1 Gerhard Bauer.1 Internal Medicine/Stem Cell Program, University of CaliforniaDavis, Sacramento, CA HIV gene therapy offers a potential alternative to current small molecule antiretroviral treatments which, after prolonged use, can become toxic and allow the generation of escape mutants Current HIV gene therapy protocols rely on ex vivo transductions of hematopoietic stem cells or peripheral blood mononuclear cells (PBMCs) These procedures require either apheresis of PBMCs, mobilization of peripheral blood stem cells, or bone marrow aspirations, methods to isolate the target cells, clinical grade tissue culture methods to introduce the vector, and finally re-administration of the gene modified cells into the patient The development of cell specific targeting vectors capable of selectively transducing cells of interest, in vivo, would greatly simplify and enhance gene therapy applications, by bringing them to areas where sophisticated laboratories and clinics are not available As a first step towards achieving in vivo transduction protocols for HIV gene therapy, a promising strategy is currently being evaluated in our laboratory Based on previous work using a Protein-A ZZ-domain/monoclonal antibody (mAb) conjugated Sindbis virus glycoprotein pseudotyped lentiviral vector, here we demonstrated the utility of this strategy for HIV gene therapy by specifically targeting HIV susceptible cells and making them resistant to HIV infection Using a CCR5 mAb conjugated envelope, CCR5 positive cells were specifically targeted for transduction by a lentiviral vector containing a highly potent CCR5 S31 ... the step of integration But the cellular response to the incoming viral protein and potential interaction between IN and cellular proteins is unknown This study reveals that HIV integrase interacts... decreased the lentiviral integration rate, while TTRAP over expression increased the integration apparently Interestingly, TTRAP facilitates the lentiviral vector integration in a PML–independent... 75 Sp100 Inhibited the Lentiviral Gene Expression Liping Qu, Jinzhong Chen Institute of Genetics, Fudan University, Shanghai, China The lentivirus integrase HIV -IN plays a crucial role in the viral