Lentiviral gene therapy for the production of systemic proteins

Abstract

Lentiviral vectors are highly effective gene transfer agents (GTAs) used to restore expression of deficient proteins in target cells. In pursuit of a GTA with efficient pulmonary transduction, the UK Respiratory Gene Therapy Consortium developed a lentivirus pseudotyped with the envelope proteins, F and HN from Sendai virus (rSIV.F/HN). In contrary to other viral vectors, pulmonary delivery of rSIV.F/HN achieves sustained gene expression (~2 years in mice) in the lungs and the systemic circulation following a single dose. Building upon previous data, the project objective was to investigate the application of the rSIV.F/HN vector-platform for wider indications, including systemic disorders and diseases that require regulated expression of a therapeutic protein. To investigate rSIV.F/HN therapy for systemic diseases, intravenous vector delivery was characterised and compared against intranasal administration, the canonical delivery route. Intranasal and intravenous vector administration achieved sustained (at least 1 year) systemic expression of secreted reporter protein, Gaussia luciferase, with one-log higher expression observed following intravenous delivery of the highest vector dose. Systemic rSIV.F/HN delivery resulted in widespread biodistribution, with the generation of significant anti-vector neutralising antibodies limiting vector re-administration. In contrast, localised airway transduction was observed following intranasal administration and maintained transduction efficiency following re-administration demonstrated previously (Chapter 3). Thrombotic thrombocytopaenic purpura (TTP), caused by deficiency of protease ADAMTS13 (a-disintegrin-and-metalloproteinase-with-a-thrombospondin-type-1-motif-13), was selected as an exemplar disease to investigate rSIV.F/HN therapy for systemic disorders. rSIV.F/HN-ADAMTS13 transduction generated dose-dependent levels of protease activity in vitro. Limited plasma expression of ADAMTS13 was achieved following delivery of rSIV.F/HN-ADAMTS13 to deficient mice, however restoration of wildtype activity levels was demonstrated in the lungs following intranasal administration (Chapter 4). To permit the application of rSIV.F/HN vectors towards indications with narrow efficacy/toxicity windows, temporal control of gene expression was investigated. Based upon a commercial yeast-progesterone system, proof-of-concept that lentivirus-mediated gene expression could be regulated was demonstrated in a human airway model and a pulmonary cell therapy model. Humanised regulated expression cassettes with yeast elements substituted for zinc finger sequences were constructed, with further investigation required to assess efficacy of humanised constructs in lentiviral vectors (Chapter 5). Data presented here supports the application of rSIV.F/HN towards systemic disorders whilst regulated expression vectors have the potential to broaden the rSIV.F/HN technology-platform for therapeutic indications that require controlled gene expression.