Publications (6)20.61 Total impact
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Article: Functional CFTR Expression in Cystic Fibrosis Airway Epithelial Cells by AAV6.2-mediated Segmental Trans-splicing.
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ABSTRACT: Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl‾ transporter. The packaging constraints of adeno-associated virus (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, where 2 AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of 2 pre-mRNAs into a full length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, co-transfection of donor and acceptor plasmids into CFTR- cells resulted in full length CFTR message and protein. Microinjection of plasmids into CFTR- cells produced a cAMP-activated Cl‾ conductance. Vectors created using an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs resulting in full length CFTR mRNA and protein as well as a cAMP-activated Cl‾ conductance in CFTR- cells including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.Human gene therapy 12/2008; · 4.20 Impact Factor -
Article: A comparative analysis of novel fluorescent proteins as reporters for gene transfer studies.
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ABSTRACT: We evaluated novel fluorescent proteins (FPs) as reporters for gene transfer in animals and cells with the aim to develop more-sensitive assays for vector development and the optimization of gene transfer strategies in gene therapy. Adeno-associated virus serotype 5 vectors carrying an expression cassette with a chicken beta-actin promoter encoding the green FPs ZsGreen1, AcGFP1, hMGFP (with and without intron), and EGFP and the red FPs DsRed2 and TurboRFP were administered to mice at identical doses for each organ to target liver, lung, and muscle. Despite the fact that all FPs were expressed from an identical vector backbone, the observed number of fluorescent cells and fluorescence intensities varied between, but was consistent within, each combination of a specific protein and organ. The highest number of fluorescent cells was observed in liver with EGFP and in lung with ZsGreen1 and EGFP. In muscle, AcGFP1 and ZsGreen1 produced the most-intense fluorescence in fibers. In contrast, in culture cells, ZsGreen1 showed substantially stronger fluorescence than all other proteins. Our data demonstrate that each FP has tissue-specific expression profiles that need to be taken into consideration when comparing the performance of vectors in different organs.Journal of Histochemistry and Cytochemistry 10/2007; 55(9):931-9. · 2.72 Impact Factor -
Article: Detection of reporter gene expression in murine airways.
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ABSTRACT: We have shown that to overcome the low levels of expression from gene transfer vector-mediated beta-galactosidase expression in lung, it is essential to replace the cytoplasmic beta-galactosidase gene with a nuclear targeted beta-galactosidase gene. We found that lung should be sectioned and fixed prior to staining for beta-galactosidase expression and that en bloc staining of intact lung is inefficient at staining positively transduced cells located deeper in the lung spaces. For GFP fluorescence, it is important to inflate the lungs with fixative prior to freezing and subsequent sectioning. For processing of nasal tissues for beta-galactosidase expression, we expand on a protocol used in previously reported gene transfer studies.Methods in molecular biology (Clifton, N.J.) 02/2007; 411:25-34. -
Article: Targeting viral-mediated transduction to the lung airway epithelium with the anti-inflammatory cationic lipid dexamethasone-spermine.
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ABSTRACT: We formulated adenovirus (AdV) vectors with cationic steroid liposomes containing dexamethasone-spermine (DS)/dioleoylphosphatidylethanolamine (DOPE) in an effort to overcome the lack of apically expressed AdV vector receptors on airway epithelial cells and to reduce the inflammation associated with AdV vector exposure. An AdV vector (1 to 2.5 x 10(11) genome copies) expressing human placental alkaline phosphatase or beta-galactosidase (LacZ) was delivered alone or complexed with DS/DOPE, DC-Chol/DOPE, or dexamethasone to C57Bl/6 mice via intranasal instillation. Formulation of the AdV vector with DS/DOPE and DC-Chol/DOPE resulted in transgene expression targeted only to the airway epithelial cells with minimal expression in alveolar cells, while AdV alone caused high alveolar transduction. The DS/DOPE and dexamethasone formulations greatly reduced cellular infiltrates compared to AdV vector alone, while formulation with DC-Chol/DOPE did not. IFN-gamma was significantly elevated at day 7 in mice receiving only the AdV vector compared to the AdV vector formulated with DS/DOPE, DC-Chol/DOPE, or dexamethasone. Lipid formulation of adeno-associated virus vector expressing LacZ also produced airway epithelial targeting, similar to the AdV vector. Viral vectors can be formulated with DS/DOPE to improve targeting to the airway epithelium in vivo and to attenuate vector-induced inflammation through the pharmacological activity of DS.Molecular Therapy 10/2005; 12(3):502-9. · 6.87 Impact Factor -
Article: An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer.
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ABSTRACT: Staining by 5-bromo-4-chloro-3-indolyl-beta-D: -galactopyranoside (X-gal) typically detects activity of E. coli beta-galactosidase (beta-gal) in transduced tissues that express the LacZ reporter gene. In lung tissue from mice that received beta-galactosidase-expressing adeno-associated virus (AAV) vectors via intranasal inhalation, we observed only a low frequency of positive cells after X-gal staining in contrast to other reporter genes, such as alkaline phosphatase or green fluorescent protein. In this study, we systematically tested a number of parameters to improve the sensitivity of X-gal staining in lungs transduced with beta-galactosidase-expressing AAV2/5 vectors. We observed that the use of nuclear-targeted LacZ instead of cytoplasmic LacZ as the reporter gene substantially increases the number of positive cells after X-gal staining. The pH of the staining solution determines staining sensitivity and background staining with pH 7.0 resulting in high sensitivity and no background levels. Glutaraldehyde at 0.2% or 0.5% in PBS as fixative provides optimal results for X-gal staining. The alternative substrate, Bluo-gal, showed no improvement compared with X-gal but instead caused nonspecific background staining. We further stained intact fixed lungs with X-gal and processed them for paraffin embedding or cryosectioning, resulting in equal staining intensities. However, en bloc staining of intact tissues resulted in the absence of positive cells within deeper-located lung areas.Histochemie 08/2005; 124(1):77-85. · 2.59 Impact Factor -
Article: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer.
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ABSTRACT: The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.Human Gene Therapy 12/2002; 13(16):1961-70. · 4.22 Impact Factor
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Institutions
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2007
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University of Pennsylvania
- Department of Pathology and Laboratory Medicine
Philadelphia, PA, USA
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2005
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Hospital of the University of Pennsylvania
- Division of General Medicine
Philadelphia, PA, USA
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