Validation of recombinant Sendai virus in a non-natural host model (original) (raw)
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Efficient gene transfer to airway epithelium using recombinant Sendai virus
Nature biotechnology, 2000
Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.
Sendai virus-mediated CFTR gene transfer to the airway epithelium
Gene Therapy, 2007
The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra-and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr G551D and Cftr tm1Unc TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.
Human Gene Therapy, 2010
Over the last two decades, enormous effort has been focused on developing virus-based gene delivery vectors to target the respiratory airway epithelium as a potential treatment for cystic fibrosis (CF) lung disease. However, amongst other problems, the efficiency of gene delivery to the differentiated airway epithelial cells of the lung has been too low for clinical benefit. Although not a target for CF therapy, the nasal epithelium exhibits cellular morphology and composition similar to that of the lower airways, thus representing an accessible and relevant tissue target for evaluating novel and improved gene delivery vectors. We previously reported that replicationcompetent human parainfluenza virus (PIV)-based vectors efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to sufficient numbers of cultured CF airway epithelial cells to completely correct the bioelectric function of CF cells to normal levels, resulting in restoration of mucus transport. Here, using an in vitro model of rhesus airway epithelium, we demonstrate that PIV mediates efficient gene transfer in rhesus epithelium as in the human counterpart. Naive rhesus macaques were inoculated intranasally with a PIV vector expressing rhesus macaque a-fetoprotein (rhAFP), and expression was monitored longitudinally. rhAFP was detected in nasal lavage fluid and in serum samples, indicating that PIV-mediated gene transfer was effective and that rhAFP was secreted into both mucosal and serosal compartments. Although expression was transient, lasting up to 10 days, it paralleled virus replication, suggesting that as PIV was cleared, rhAFP expression was lost. No adverse reactions or signs of discomfort were noted, and only mild, transient elevations of a small number of inflammatory cytokines were measured at the peak of virus replication. In summary, rhAFP proved suitable for monitoring in vivo gene delivery over time, and PIV vectors appear to be promising airway-specific gene transfer vehicles that warrant further development.
The Journal of Gene Medicine, 2013
Background-A limitation to efficient lentivirus-mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. Methods-We investigated influenza hemagglutinin (HA) pseudotyped EIAV-derived lentiviral vector-mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies high-titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re-dosing to the nasal airway. Results-Delivery of high-titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re-dosing to the nasal airway of mice was found to be dependent upon the age of the animal when the first dose is administered and the length of time between doses. Conclusions-A single intranasal dose of concentrated influenza HA-pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases.
Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors
Molecular therapy. Nucleic acids, 2013
Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better ...
Virus Research, 2006
Sendai virus (SeV) and human parainfluenza virus type I (hPIV1) are highly homologous but have distinct host ranges, murine versus human. To identify the factors that affect the host specificity of parainfluenza viruses, we determined the infectivity and anti-IFN activities of SeV and hPIV1 in human and murine culture cells. SeV infected normal human lung MRC-5 and murine lung MM14.Lu or MLg2908 cells efficiently. Infection with SeV induced the release of IFN- into culture medium in MRC-5 cells at similar levels with that of cells infected with hPIV1. SeV or hPIV1 infections, as well as expression of SeV or hPIV1 C proteins, inhibited the nuclear localization of STAT1 induced by IFN-, suggesting that both SeV and hPIV1 C proteins block the IFN Jak/STAT pathway in MRC-5 cells. Pretreatment of MRC-5 cells with IFN suppressed replication of SeV and hPIV1 at an early stage of infection. However, hPIV1 overcame this suppression while SeV did not. SeV replication was restored in IFN- pretreated murine MM14.Lu cells, suggesting SeV anti-IFN activity is species specific. These results suggest that SeV is less effective than hPIV1 in overcoming antiviral activity in human cells, which could be one of the factors that restrict the host range of SeV.
Anti-Cancer Drugs, 2008
Few studies have directly compared the efficiencies of gene delivery methods that target normal lung cells versus lung tumor cells. We report the first study directly comparing the efficiency and toxicity of viral [adenoassociated virus (AAV2, 5, 6) and lentivirus], nonviral (Effectene, SuperFect and Lipofectamine 2000) and physical [particle-mediated gene transfer (PMGT)] methods of gene delivery in normal mouse lung cells and in mouse adenocarcinoma cells. Lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein was the most efficient gene transfer method for normal mouse airway epithelial cells [25.95 ( ± 3.57) %] whereas AAV6 was most efficient for MLE-12 adenocarcinoma cells [68.2 ( ± 3.2) %]. PMGT was more efficient in normal mouse airway epithelial cells than AAV5, Lipofectamine 2000 and SuperFect. AAV5 displayed the lowest transfection efficiency at less than 10% in both cell types. PMGT was the only method that resulted in significant toxicity. In summary, for all of the gene delivery methods examined here, lung tumor cells were transfected more easily than normal lung cells. Lipofectamine 2000 is potentially highly selective for lung tumor cells whereas AAV6 and lentivirus vesicular stomatitis virus glycoprotein may be useful for gene delivery strategies that require targeting of both normal and tumor cells.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2013
Background-A limitation to efficient lentivirus-mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. Methods-We investigated influenza hemagglutinin (HA) pseudotyped EIAV-derived lentiviral vector-mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies high-titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re-dosing to the nasal airway. Results-Delivery of high-titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re-dosing to the nasal airway of mice was found to be dependent upon the age of the animal when the first dose is administered and the length of time between doses. Conclusions-A single intranasal dose of concentrated influenza HA-pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases.