Investigating pseudotyped lentiviral vectors for gene delivery to the lung

Thesis

Recombinant lentiviral vectors (LV) are an attractive vehicle for gene delivery to the lung due to their ability to achieve stable, long-term, expression and the opportunity to add glycoprotein pseudotypes for efficient lung cell-targeting. Two LV pseudotypes derived from sialic acid- binding Sendai virus and influenza virus, referred to as F/HN and Rostock respectively, can facilitate efficient in vivo transduction of the murine airway and were compared alongside a new influenza-based pseudotype (Shanghai) which, in contrast to F/HN and Rostock, is based on a strain that infects humans. Lectin staining confirmed a sialylated receptor distribution that is different between murine and human lung, with a greater abundance of a2,6 and relative lack of an a2,3 subtype in the human airway, such that transduction of mouse lung is likely a poor predictor of performance in humans. Ex vivo transduction of murine precision cut lung slices (PCLS) recapitulated results from in vivo experiments. However, PCLS from resected human lung did not contain airways, and therefore cultures of human airway epithelial cells, maintained at an Air-Liquid Interface (ALI), were used to model transduction of the polarised airway epithelium. Different cellular source materials and ALI culture protocols showed differences in the abundance of human sialylated receptor subtypes available, as well as subsequent transduction efficiency by F/HN pseudotyped LV. Importantly, transduction of human ALI cultures was more efficient using vectors based on human as opposed to simian immunodeficiency virus. A greater transduction efficiency was observed for Shanghai pseudotyped LV compared with Rostock but, overall the F/HN pseudotype demonstrated superior efficiency, transducing all human airway cell types characterised. This was consistent with the greater affinity of the F/HN pseudotype for the a2,3 sialylated (N-acetyllactosamine) receptor present in all models investigated, and also explained the broad range of pulmonary cell-types (including the repopulating/progenitor cells of murine airway) transduced in all models investigated. These data support the strong translational potential of the F/HN pseudotype.

Authors: Munday, RJ

• DOI: 10.5287/ora-kebrv0rx8
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: pseudotyping; Sendai virus; gene therapy; neuraminidase; air-liquid interface; airway; hemagglutinin; lentiviral vector; lung; influenza virus; sialylated receptor
• Subjects: Lentiviruses; Sendai virus; Transduction; Avian influenza A virus; Lungs; Gene therapy