Sabrina Gea-Sorli

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air–liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and ‘benchmarked’ against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90–100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.

S56 Moving lentiviral-based gene therapy into a first-in-man CF trial

The UK CF Gene Therapy Consortium has developed a pipeline of vectors to deliver CFTR into the airway epithelium. The first of these (plasmid/liposome complexes) recently completed a Phase IIb trial. Anticipating that increased efficiency of gene transfer will be required, we have developed an F/HN-pseudotyped lentivirus which is ~2 logs more efficient in lung gene transfer than non-viral vectors, a single administration lasts for the lifetime of a mouse, and can be repeatedly administered. This vector is targeted for a first-in-man study in 2016, and in preparation for this we have assessed (1) selection of the most efficient promoter/enhancer for lung gene transfer, (2) assessment of toxicity “benchmarked” against the leading non-viral formulation including mapping of integration sites, (3) determination of transduction efficiency which will be used to inform dose-ranging in the trial and characterisation of the cell types transduced by the vector, (4) understanding the impact of pre-existing and acquired anti-viral immunity on transduction efficiency and toxicity, (5) confirmation of CFTR expression and function in relevant models and (6) comparison of vector stability in a jet and single-pass mesh nebuliser. Data will be presented for each of these components, which we believe support progression into human studies. Trial design as well as a regulatory-compliant toxicology study will also be discussed.

DEVELOPMENT OF AN OPTIMAL F/HN PSEUDOTYPED SIV VECTOR FOR CF GENE THERAPY

We are developing lung gene transfer vectors to treat acquired and inherited lung disorders such as cystic fibrosis, and have identified two platforms for efficient respiratory gene delivery: one non-viral system based on CpG-free plasmid DNA combined with cationic lipids (pDNA/GL67A), which has recently completed evaluation in a Phase IIb clinical study; and one novel viral system based on a recombinant simian immunodeficiency virus pseudotyped with the F/HN proteins of Sendai virus (rSIV. F/HN) to promote airway cell uptake. Here we report on the development of a “third generation” rSIV. F/HN vector suitable for use in the clinic. The vector is manufactured by transient transfection of cultured human cells using five producer plasmids, all of which have been engineered to be pharmacopoeia compliant. A variety of vector configurations, including a range of enhancers/promoters and transgenes, were evaluated in a panel of airway models. rSIV. F/HN vectors directed high-level, robust gene expression in fully differentiated human airway cells, human nasal brushings and human and sheep lung slices. In the mouse nose and lung, the preferred configuration directed up to x500-fold higher transgene expression than the non-viral platform, for the lifetime of the animal. Transgene expression was observed in 14.1% of lung epithelial cells (p < 0.0001 compared to controls). Repeated monthly administration (3X) was 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 rSIV. F/HN 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). Delivery of rSIV. F/HN expressing CFTR to sheep lung resulted in CFTR mRNA at ∼30% the levels of endogenous ovine CFTR (p < 0.0001 compared to non-treated lobes), exceeding presumed therapeutic levels. With the majority of translational hurdles addressed, we are now entering toxicology studies and the final stages of pharmaceutical development prior to entering clinical trials.