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.