Molecular Therapy, Vol 22, S1, Abstract 372
The American Society of Gene and Cell Therapy Annual Conference, Washington DC, 2014
We are developing a lentiviral gene transfer vector to treat CF lung disease. Our preferred F/HN-SIV vector is based on simian immunodeficiency virus (SIV), pseudotyped with the F/HN proteins of Sendai virus to promote efficient airway cell uptake. Previously we have shown that a single dose of 2nd generation F/HN-SIV results in lung reporter expression for the lifetime of the mouse (∼2yrs) and that repeat (3X) monthly administration was possible without loss of efficacy. We have now developed an optimal 3rd generation F/HN-SIV vector expressing CFTR suitable for use in the clinic. The 5 constituent plasmids were engineered to be pharmacopoeial compliant.
A variety of vector configurations, including a range of enhancers/promoters and transgenes, were evaluated in airway models. F/HN-SIV vectors directed high-level, robust reporter gene expression in fully differentiated human airway cells, human nasal brushings and human and sheep lung slices. In the mouse nose and lung, F/HN-SIV vectors directed abundant, stable luciferase reporter gene expression for the duration of all in vivo studies (study length: 1-7 months). At day 14 in the mouse lung, F/HN-SIV GFP vectors directed reporter gene expression in 14.1% of lung epithelial cells (P<0.0001 compared to controls). Repeated monthly administration of these vectors (3X) was also possible without loss of expression or significant histological inflammatory reactivity.
Reassuringly, insertion site profiling from transduced cell lines and mouse nose/lung samples reveals a pattern of integration comparable to those reported for other lentiviral vectors in clinical development, with no evidence for enrichment of insertion at undesirable loci. The stability of F/HN-SIV vectors was evaluated in two bronchoscope catheters and two aerosol generation devices.
Encouragingly for clinical translation, no significant loss of transduction activity was noted with any of these clinically relevant delivery devices (P=0.64). In large animal studies, delivery of F/HN-SIV expressing CFTR to sheep lung lobes by bronchoscopic instillation resulted in vector-derived CFTR mRNA at ∼30% the levels of endogenous ovine CFTR in the treated lobes at day 7 (P<0.0001 compared to non-treated lobes), exceeding presumed therapeutic levels. To catalyse the development of a large-scale cGMP-compatible virus production methodology, we have developed a novel approach based on scalable serum-free suspension cell culture in rocking bioreactors. This is animal-free, and utilises cGMP-compliant virus capture and purification reagents.
Median purified vector titres exceed 2e9 TU/mL. This process has been approved by the UK MHRA for regulatory studies, and can be scaled for our planned toxicology studies and early clinical trials. With the majority of translational hurdles addressed, we are now entering the final stages of development of our F/HN-SIV vector system prior to entering clinical trials.