Dual reporter comparative indexing of rAAV pseudotyped vectors in chimpanzee airway.

Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Molecular Therapy (Impact Factor: 7.04). 10/2009; 18(3):594-600. DOI: 10.1038/mt.2009.230
Source: PubMed

ABSTRACT Selecting the most efficient recombinant adeno-associated virus (rAAV) serotype for airway gene therapy has been difficult due to cross-specific differences in tropism and immune response between humans and animal models. Chimpanzees--the closest surviving genetic relative of humans--provide a valuable opportunity to select the most effective serotypes for clinical trials in humans. However, designing informative experiments using this protected species is challenging due to limited availability and experimental regulations. We have developed a method using Renilla luciferase (RL) and firefly luciferase (FL) reporters to directly index the relative transduction and immune response of two promising rAAV serotypes following lung coinfection. Analysis of differential luciferase activity in chimpanzee airway brushings demonstrated a 20-fold higher efficiency for rAAV1 over rAAV5 at 90 days, a finding that was similar in polarized human airway epithelia. T-cell responses to AAV5 capsid were stronger than AAV1 capsid. This dual vector indexing approach may be useful in selecting lead vector serotypes for clinical gene therapy and suggests rAAV1 is preferred for cystic fibrosis.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Human bocavirus virus-1 (HBoV1), a newly discovered autonomous parvovirus with a 5,500 nt genome, efficiently infects human-polarized airway epithelia (HAE) from the apical membrane. We hypothesized that the larger genome and high airway tropism of HBoV1 would be ideal for creating a viral vector for lung gene therapy. To this end, we successfully generated recombinant HBoV1 (rHBoV1) from an open reading frames-disrupted rHBoV1 genome that efficiently transduces HAE from the apical surface. We next evaluated whether HBoV1 capsids could package oversized rAAV2 genomes. These studies created a rAAV2/HBoV1 chimeric virus (5.5 kb genome) capable of apically transducing HAE at 5.6- and 70-fold greater efficiency than rAAV1 or rAAV2 (4.7-kb genomes), respectively. Molecular studies demonstrated that viral uptake from the apical surface was significantly greater for rAAV2/HBoV1 than for rAAV2 or rAAV1, and that polarization of airway epithelial cells was required for HBoV1 capsid-mediated gene transfer. Furthermore, rAAV2/HBoV1-CFTR virus containing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene coding sequence and the strong CBA promoter efficiently corrected CFTR-dependent chloride transport in cystic fibrosis (CF) HAE. In summary, using the combined advantages of AAV and HBoV1, we have developed a novel and promising viral vector for CF lung gene therapy and also potentially HBoV1 vaccine development.Molecular Therapy (2013); doi:10.1038/mt.2013.92.
    Molecular Therapy 07/2013; · 7.04 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Idiopathic pulmonary arterial hypertension (iPAH) is associated with high morbidity and mortality. We evaluated whether luminal delivery of the human prostacyclin synthase (hPGIS) cDNA with adenoassociated virus (AAV) vectors could attenuate PAH. AAV serotype 5 and 9 vectors containing the hPGIS cDNA under the control of a cytomegalovirus (CMV)-enhanced chicken β-actin promoter (CB) or vehicle (saline) were instilled into lungs of rats. Two days later, rats were injected with monocrotaline (MCT, 60 mg/kg) or saline. Biochemical, hemodynamic, and morphologic assessments were performed when the rats developed symptoms (3-4 weeks) or at six weeks. Luminal (airway) administration of AAV5 and AAV9 CBhPGIS vectors (MCT-AAV5 and MCT-AAV9 rats) significantly increased plasma levels of 6-keto-PGF1α as compared to MCT-controls, and closely resembled levels measured in rats not treated with MCT (saline-saline). Right ventricular (RV)/left ventricular (LV) + septum (S) ratios and RV systolic pressure (RVSP) were greater in MCT-control rats than in saline-saline rats, whereas the ratios and RVSP in MCT- AAV5CBhPGIS and MCT-AAV9CBhPGIS rats were similar to saline-saline rats. Thickening of the muscular media of small pulmonary arteries of MCT-control rats was detected in histological sections, whereas the thickness of the muscular media in MCT-AAV5CBhPGIS and MCT-AAV9CBhPGIS rats was similar to saline-saline controls. In experiments with different promoters, a trend toward increased levels of PGF1α expression was detected in lung homogenates, but not plasma, of MCT-treated rats transduced with an AAV-hPGIS vector containing a CB promoter. This correlated with significant reductions in the RV/LV+S ratio and RVSP in MCT-AAV9CBhPGIS rats that resembled levels in saline-saline rats. No changes in levels of PGF1α, RV/LV+S, or RVSP were detected in rats transduced with AAV9-hPGIS vectors containing a modified CB promoter (CB7) or a distal epithelial cell specific promoter (CC10). Thus, AAV9CBhPGIS vectors prevented development of MCT-induced PAH and associated pulmonary vascular remodeling.
    Human gene therapy 02/2014; · 4.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We recently created a cystic fibrosis (CF) ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated rAAV-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret polarized airway epithelia (FAE), in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were due to in vivo secreted factors that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor(s) included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that post-entry processing of virus was influenced by the inhibitor factor(s). Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that factor(s) in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.
    Human gene therapy 08/2013; · 4.20 Impact Factor


Available from
May 20, 2014