Mc Paul-Smith

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.

Gene therapy for cystic fibrosis: recent progress and current aims

ABSTRACT Introduction: Since identification of the disease causing gene over 25 years ago, cystic fibrosis (CF) has been at the forefront of gene therapy research. Despite initial optimism, CF gene therapy has proven considerably more challenging than initially anticipated. However, research conducted over the past two decades has clarified the strength and weaknesses of viral and non-viral gene transfer agents for CF gene therapy. Areas covered: The older literature related to CF gene therapy has been reviewed in many publications and we will, therefore, restrict this review to a brief description and discussion of the key lessons learnt, instead focusing on more recent progress in the field which was identified through literature searches. This review will summarize research leading up to the recent pivotal proof-of-concept study showing that non-viral gene therapy can stabilize the decline of lung function in CF patients and also highlight recent advances in viral vector development which may overcome problems related to loss of efficacy on repeated administration. Expert opinion: The demonstration that gene therapy can stabilize CF lung disease is an important milestone in gene therapy.

The murine lung as a factory to produce secreted intrapulmonary and circulatory proteins

We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 μg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.

RSIV. F/HN-MEDIATED GENE THERAPY ENABLES LUNGS TO PRODUCE THERAPEUTICALLY RELEVANT LEVELS OF FVIII

We have previously shown that lung when treated with Sendai virus-mediated gene transfer can produce secreted proteins and release them into the circulation (Griesenbach et al., Mol Therapy 2002). Despite the high levels of transduction efficiency the gene expression is transient and repeated administration is not feasible due to induction of immune responses. To overcome these barriers we developed a lentiviral vector specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV. F/HN) to allow efficient transduction of the airways. Stable expression for >20 months after a single dose and efficient transduction after repeated administration despite detection of anti-rSIV. F/HN neutralising antibodies make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV. F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n = 5 –6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p < 0.01) levels of GLux in serum (274 ± 72 RLU/ul, control: 41 ± 6 RLU/ul) which persisted for at least 12 months further supporting the hypothesis that the lung is a suitable, non-invasive factory for production of secreted proteins. Gene therapy strategies for haemophilia have focussed on intravenous or intramuscular delivery of the gene transfer agent. Here, we treated the murine lung with rSIV. F/HN carrying the FVIII cDNA (1.6e8–3.4e8 TU/mouse,) or placebo and assessed whether therapeutically relevant levels of FVIII can be produced. Significant (p < 0.05) and dose-related levels of FVIII were detectable in lungs and BALF 10 and 28 days post-transduction. Dose-related levels of FVIII were also detectable in plasma, which reached a therapeutically relevant level of 3% of normal 1 month after gene transfer. These data support the concept that rSIV. F/HN-mediated transduction of lungs can produce therapeutically relevant and persistent levels of recombinant protein in blood. Reference 1 Griesenbach U, Cassady RL, Ferrari S et al. The nasal epithelium as a factory for systemic protein delivery. Mol Ther. 2002;5:98–103

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.

S127 Gene therapy for alpha-1-antitrypsin deficiency using a pseudotyped lentivirus vector

Introduction and objectives A protease/anti-protease imbalance is a characteristic feature of inflammatory lung diseases such as cystic fibrosis (CF) and alpha-1-antitrypsin deficiency related emphysema. A recent trial of alpha-1-antitrypsin (hAAT) enzyme replacement therapy (ERT) suggested that hAAT can slow the progression of lung density loss in alpha-1-antitrypsin deficiency (Chapman et al, Lancet 2015). However, the results are modest and ERT is expensive, so gene therapy may be a more appropriate treatment strategy. The UK Cystic Fibrosis Gene Therapy Consortium has pseudotyped a simian immunodeficiency viral vector with the Sendai virus F and HN proteins (rSIV. F/HN) for efficient transduction of airway epithelial cells. Results Mice were transduced with rSIV. F/HN-hAAT (1.4e8 TU/mouse) by nasal instillation and culled 10 days post-transduction. hAAT levels in lung tissue homogenate and epithelial lining fluid (ELF) were 3 logs above controls (p < 0.05), and hAAT concentration in ELF was 92 ± 28 μg/ml, similar to the therapeutic hAAT level in ELF of 70 μg/ml (Figure 1). For comparison, transfection of mouse lung with cationic lipid GL67A, used in the recent Phase IIb trial of non-viral gene therapy for cystic fibrosis, complexed to plasmids carrying hAAT only led to 0.4 ± 0.1 μg/ml in ELF.Abstract S127 Figure 1 Expression of hAAT in epithelial lining fluid following treatment with rSIV. F/HN-hAAT. Mice were given between 2e7 and 1.4e8 TU virus and sacrificed 7–10 days post-treatment A neutrophil elastase (NE) activity assay showed that the recombinant hAAT successfully neutralised NE activity (p < 0.05). In a separate experiment, mice were treated with a single dose of rSIV. F/HN-hAAT (4e7 TU/mouse) and quantification of hAAT one year post-transduction showed that expression was stable over this period. Here, we also demonstrate for the first time that rSIV. F/HN transduction of lung generates significant (p < 0.05) levels of recombinant hAAT protein in serum. Conclusion In conclusion, rSIV. F/HN produces therapeutically relevant and long-lasting levels of hAAT in murine lung and may offer advantages over enzyme replacement therapy. In addition, we showed that hAAT escapes from the lung into the circulation which may be relevant for a range of diseases including diabetes and graft vs. host disease. Reference 1 Chapman KR, Burdon JG, Piitulainen E et al. Intravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial. Lancet. 2015;386:360–8

171. Production of Therapeutically Relevant Levels of FVIII After Transduction of Lungs With F/HN-Pseudotyped Lentivirus

We have previously shown that the lung can be used as “factory” for production of secreted proteins, which are released into the circulation, using Sendai virus-mediated gene transfer. However, gene expression was transient and repeated administration of the vector was not feasible due to induction of immune responses. We have developed a lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN) to allow efficient transduction of the airways. The unique features of rSIV.F/HN, namely stable expression for >20 months after a single dose and efficient transduction after repeated administration, make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV.F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n=5-6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p<0.01) levels of GLux in serum (274±72 RLU/ul, control: 41±6 RLU/ul) which persisted for at least 12 months further supporting the hypothesis that the lung is a suitable, non-invasive factory for production of secreted proteins. Gene therapy strategies for haemophilia have focussed on intravenous or intramuscular delivery of the gene transfer agent. Here, we treated the murine lung with rSIV.F/HN carrying the FVIII cDNA (1.6e8-3.4e8 TU/mouse, n=3-4/group) or placebo and assessed whether therapeutically relevant levels of FVIII can be produced. Significant (p<0.05) and dose-related levels of FVIII were detectable in lungs and BALF 10 and 28 days post-transduction. Dose-related levels of FVIII were also detectable in plasma, which reached a therapeutically relevant level of 3.0% of normal 1 month after gene transfer. These data support the concept that rSIV.F/HN-mediated transduction of lungs can produce therapeutically relevant and persistent levels of recombinant protein in blood.

370. F/HN Pseudotyped Lentivirus Generates Therapeutically Relevant and Long-Lasting Alpha-1-Antitrypsin Expression in Mouse Lung

A protease/anti-protease imbalance is a characteristic feature of inflammatory lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). However, alpha-1-antitrypsin (AAT) enzyme replacement therapy (ERT) trials have not shown conclusive evidence of a therapeutic benefit. This may be due to inefficient protein delivery to the lung and/or the half-life of the protein. The UK Cystic Fibrosis Gene Therapy Consortium (GTC) has pseudotyped a simian immunodeficiency viral vector with the Sendai virus F and HN proteins (rSIV.F/HN) and selected an internal synthetic promoter/enhancer consisting of the elongation factor 1α promoter and the CMV enhancer (hCEF) for efficient transduction of airway epithelial cells. Here, we assessed whether transduction of murine lungs with rSIV.F/HN with a human AAT transgene generates therapeutic levels of AAT. Mice were transduced with rSIV.F/HN-hCEF-AAT (2e8 TU/mouse) by nasal instillation and culled 10 days after transduction. AAT levels in lung homogenate and broncho-alveolar lavage fluid (BALF) were at least 3 logs above controls (p<0.05). A urea assay was used to determine that lavage diluted the epithelial lining fluid (ELF) by ≈40-fold, which allowed us to calculate the AAT concentration in ELF to be 92±28 mg/ml (= 1.4 μμ). In the context of AAT deficiency, an ELF concentration of 70 mg/ml (1.1 μM) AAT is of therapeutic relevance. For comparison transfection of mouse lung with the cationic lipid GL67A complexed to pCMV-AAT only led to 0.4±0.1 mg/ml (n=6) of AAT in ELF. A neutrophil elastase (NE) activity assay showed that the recombinant AAT successfully neutralised NE activity (p<0.05). In a separate experiment, mice were treated with rSIV.F/HN-hCEF-AAT (4e7 TU/mouse) and quantification of AAT 7 and 90 days post-transduction showed that expression was stable over this period. We also transduced mice with rSIV.F/HN-hCEF carrying a secreted Gaussia luciferase reporter gene (1e7 TU/mouse) and showed that stable expression in lung and BALF persisted for at least 12 months. Here, we also demonstrate for the first time that rSIV.F/HN transduction of lung generates significant (p<0.05) levels of GLux and AAT recombinant proteins in serum. AAT enzyme replacement therapy is currently being assessed for the treatment of a range of diseases including diabetes and graft-vs-host disease, but shortage of human plasma-derived protein, as well as high costs, may limit the application. In conclusion, rSIV.F/HN produces therapeutically relevant and long lasting levels of AAT in murine lung. In addition, we showed that AAT escapes from the lung into the circulation, suggesting that gene therapy may help to overcome some of the current bottlenecks of ERT.