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Ze-Yan Yu,
Karen McKay,
Peter van Asperen,
Maolin Zheng, Jane Fleming,
Samantha L Ginn,
Eddy Kizana,
Margot Latham,
Michael P Feneley,
Peter D Kirkland,
Peter B Rowe,
Eugenie R Lumbers,
Ian E Alexander
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ABSTRACT: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges.
In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway.
We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low.
This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.
The Journal of Gene Medicine 07/2007; 9(6):429-39. · 2.48 Impact Factor
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ABSTRACT: Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.
Human Gene Therapy 09/2005; 16(8):947-56. · 4.22 Impact Factor
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ABSTRACT: Molecular Therapy (2005) 11, S188|[ndash]|S188; doi: 10.1016/j.ymthe.2005.07.025
485. Lentivirus-Mediated Gene Transfer to Cultured Fetal Sheep Airway Epithelium and Lung Tissue Explants
Ze-Yan Yu1,2,|[ast]|, Karen McKay1, Peter Van Asperen1, Maolin Zheng2, Jane Fleming2, Samantha Ginn2, Eugenie Lumbers3, Peter Rowe2 and Ian Alexander21Respiratory Medicine, The Children's Hospital at Westmead, Sydney, NSW, Australia2Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Sydney, NSW, Australia3School of Medical Sciences, University of NSW, Sydney, NSW, Australia|[ast]|ZYY was supported by the Flame Opals Foundation.
Molecular Therapy 04/2005; · 6.87 Impact Factor
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ABSTRACT: In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency virus type 1 (HIV-1) accessory proteins. Cell-type specificity of CMV promoter expression, tropism of the VSV-G envelope, and blocks to molecular transduction were also precluded as possible mechanisms, thereby implicating transcriptional repression of the internal heterologous promoter. This promoter interference effect was found to be mediated by cis-acting sequences upstream of the core promoter elements located in the U3 region of the proviral long terminal repeats (LTRs). Deletion of this region, as in late-generation self-inactivating (SIN) lentivirus vectors, relieves this effect. This provides a basis for reevaluating data produced using early-generation U3-bearing lentivirus vectors and for reconciling these with results obtained using more contemporary SIN lentivirus vectors carrying a U3 deletion.
Human Gene Therapy 09/2003; 14(12):1127-37. · 4.22 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/2003; 229:155-67.
