Rationale and objective Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CFTR modulators offer significant improvements, but similar to 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimised for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. Methods Air-liquid interface cultures of primary human bronchial epithelial cells (HBECs) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalised human lung epithelial cell line mimicking class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. Results rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence <= 10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalisation assay. Conclusions The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

We have developed a third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) as a gene therapy for cystic fibrosis (BI 3720931). Here, we assessed transduction efficiency and acute toxicology of the rSIV.F/HN vector expressing an enhanced green fluorescent protein (EGFP) reporter gene in non-human primates (NHPs). Intubated male cynomolgus monkeys received one aerosolized dose of vector (n = 3) or placebo (n = 3). Toxicology was assessed by histopathology, clinical pathology, cytokine levels, and changes in body and organ weight. Transduction efficiency was quantified by EGFP immunohistochemistry in airway epithelial cells and vector-specific mRNA and DNA in the lung 7 days post-dosing. There were no vector-related clinical observations, mortalities, or changes in body or organ weight. Clinical pathology and cytokine analyses were unremarkable. Minimal mixed-cell centriacinar inflammation was observed in 1/3 vector-treated animals. Airway epithelial cell transduction efficiency was 9%-12%. Genomic DNA vector integration was detected in 6.7% of lung epithelial cells. Vector-specific mRNA levels were ∼45× endogenous CFTR mRNA levels in lung epithelium and ∼16× in bronchial brushings. This study extends earlier findings of rSIV.F/HN-based in vivo gene transfer in mice to NHPs, demonstrating transduction efficiency without relevant toxicity.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Although CF is a multiorgan disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with CF (estimated at 10-15% of the global CF population) who are genetically ineligible for, or intolerant of, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with CF, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides, and gene editing, being explored. Various nonviral and viral vectors have been investigated for CF gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively redosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of CF. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

Objectives: We have developed a lentiviral vector platform pseudotyped with the Sendai virus F and HN envelope proteins (rSIV.F/HN), including the clinical candidate BI 3720931 for cystic fibrosis (CF) gene therapy. We have previously demonstrated restoration of CFTR function in CF-patient bronchial epithelial cell air–liquid interface cultures and intestinal organoids, as well as efficient and persistent in vivo transduction of murine airways. We have now assessed transduction efficiency and acute toxicology in non-human primates (NHPs). Methods: Male cynomolgus monkeys received a single dose of aerosolised rSIV.F/HN vector expressing green fluorescent protein (GFP) (4.2e9 transduction units in ~2 ml) or placebo via an endotracheal tube (n = 3/group). Approximately 25% of the dose (mass median aerodynamic diameter 3.9 µm) was deposited in the lungs. Toxicology was assessed by histopathology, clinical pathology, cytokine levels and changes in body and organ weight, and transduction efficiency was quantified by GFP immunohistochemistry and vector-specific mRNA 7 days post-dosing. Results: There were no vector-related clinical observations, mortality, or changes in body or organ weight. Clinical pathology and cytokine analyses were unremarkable. A minimal mixed-cell centriacinar inflammation was observed in 1/3 active treated animals. Airway epithelial cell transduction efficiency was 9–12% and vector-specific mRNA levels were ~45x higher than endogenous CFTR mRNA levels. Conclusions: This study confirms and extends our previous findings of rSIV.F/HN-based in vivo gene transfer in mice to NHPs. Using a relatively low vector dose, NHPs demonstrated transduction efficiency in the range of values likely to relate to clinical benefit, without evidence of toxicity. Transduction efficiency and toxicology in animals treated with a higher dose is currently being analysed. These data, together with our previous murine data, support further progression of BI 3720931 towards the clinic.

PURPOSE OF REVIEW:Advances in cystic fibrosis (CF) therapies over the past decade pivotally changed the morbidity and mortality of CF with the advent of cystic fibrosis transmembrane conductance regulator (CFTR) modulators that rescue dysfunctional CFTR protein in individuals with eligible genotypes. However, a significant proportion of the CF population is in need of alternative treatment strategies to address CFTR variants that are ineligible for therapeutic protein correction and/or potentiation. Current drug development efforts of nucleic-acid based therapies (i.e., DNA and RNA based therapies) in CF are informed by historic challenges of CF gene therapy trials, recent FDA guidance informed by non-CF gene therapy trials, and advances in therapeutic applications related to severe acute respiratory syndrome coronavirus 2 vaccine development. These historic and timely developments are of significant relevance for advancing genetic therapies in CF. RECENT FINDINGS:This article reviews the main themes of semi-permanent genetic therapy strategies covering recent literature focused on: adenovirus and adeno-associated virus vector delivery, advances in lentivirus vector use and safety considerations, mRNA delivery and antisense oligonucleotide drug development. SUMMARY:Currently, drug development and clinical trials for genetic therapies in CF are rapidly progressing. This review aims to increase the foundational knowledge of CF genetic therapies.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

Human Gene TherapyVol. 31, No. 17-18 CommentaryGene Therapy for Respiratory Diseases: Progress and a Changing ContextEric W.F.W. Alton, A. Christopher Boyd, Jane C. Davies, Deborah R. Gill, Uta Griesenbach, Tracy E. Harman, Stephen Hyde, and Gerry McLachlanEric W.F.W. Alton*Correspondence: Prof. Eric W.F.W. Alton, Department of Gene Therapy, Imperial College at the National Heart and Lung Institute, Manresa Road, SW3 6LR London, United Kingdom. E-mail Address: e.alton@imperial.ac.ukGene Therapy Group, National Heart and Lung Institute, Imperial College London, London, United KingdomUK CF Gene Therapy Consortium, London, United KingdomSearch for more papers by this author, A. Christopher BoydUK CF Gene Therapy Consortium, London, United KingdomCentre for Genomic and Experimental Medicine, IGMM, University of Edinburgh, Edinburgh, United KingdomSearch for more papers by this author, Jane C. DaviesGene Therapy Group, National Heart and Lung Institute, Imperial College London, London, United KingdomUK CF Gene Therapy Consortium, London, United KingdomSearch for more papers by this author, Deborah R. GillUK CF Gene Therapy Consortium, London, United KingdomNuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United KingdomSearch for more papers by this author, Uta GriesenbachGene Therapy Group, National Heart and Lung Institute, Imperial College London, London, United KingdomUK CF Gene Therapy Consortium, London, United KingdomSearch for more papers by this author, Tracy E. HarmanGene Therapy Group, National Heart and Lung Institute, Imperial College London, London, United KingdomUK CF Gene Therapy Consortium, London, United KingdomSearch for more papers by this author, Stephen HydeUK CF Gene Therapy Consortium, London, United KingdomNuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United KingdomSearch for more papers by this author, and Gerry McLachlanUK CF Gene Therapy Consortium, London, United KingdomThe Roslin Institute & R(D)SVS, Easter Bush Campus, University of Edinburgh, Edinburgh, United Kingdom.Search for more papers by this authorPublished Online:16 Sep 2020https://doi.org/10.1089/hum.2020.142AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byFundamental and translational research in Cystic Fibrosis – why we still need itJournal of Cystic Fibrosis, Vol. 22Gene transfection using branched cationic amphiphilic compounds for an aerosol administration in cystic fibrosis contextInternational Journal of Pharmaceutics, Vol. 631CFTR RNA- and DNA-based therapiesCurrent Opinion in Pharmacology, Vol. 65Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes23 December 2021 | Pharmaceutics, Vol. 14, No. 1 Volume 31Issue 17-18Sep 2020 InformationCopyright 2020, by Mary Ann Liebert, Inc., publishersTo cite this article:Eric W.F.W. Alton, A. Christopher Boyd, Jane C. Davies, Deborah R. Gill, Uta Griesenbach, Tracy E. Harman, Stephen Hyde, and Gerry McLachlan.Gene Therapy for Respiratory Diseases: Progress and a Changing Context.Human Gene Therapy.Sep 2020.911-916.http://doi.org/10.1089/hum.2020.142Published in Volume: 31 Issue 17-18: September 16, 2020Online Ahead of Editing: August 4, 2020PDF download

The UK CF Gene Therapy Consortium has previously demonstrated that repeated delivery of a CFTR-liposome (GL67A) complex can stabilise lung function in a double-blind placebo-controlled Phase 2b trial. However, the magnitude of benefit did not warrant continued progression in the context of the welcome benefit provided by small molecule modulators. We have, in parallel, developed a Simian Immunodeficiency Virus (SIV)-based lentiviral vector pseudotyped with the Sendai-virus envelope glycoproteins (F/HN). In preclinical studies we have shown that: This is considerably more effective at transducing the respiratory epithelium than the clinically benchmarked non–viral GL67A formulation. Specifically, we observe >15% of target cells transduced in vivo in multiple species This transduction is maintained after three repeated applications Expression is maintained for up to the lifetime of a mouse from a single application We see no evidence for acute toxicity in comparison to GL67A, nor integration site hotspots or clonal expansion To prepare for a first-in-human clinical trial, we have partnered this product with Boehringer Ingelheim and Oxford BioMedica and are moving rapidly through the preparatory steps including: Developing manufacturing at scale sufficient to support a combined nasal and pulmonary Phase 1/2a study Establishing toxicology protocols suitable for both murine and higher species studies Assessing extra-pulmonary biodistribution and shedding potential Developing single cell transduction assays that can be used in both preclinical studies and the clinical trial Focusing on the initial trial population which will predominantly be recruited from the ~15% of people with CF with unmet need The above data will be discussed in the context of delivering a gene therapy trial against a background of people with CF, increasingly treated with small molecule modulators.

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.

Background Cystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco ® , Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediated CFTR gene therapy formulation through preclinical and clinical development. Objective To determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF. Design This was a randomised, double-blind, placebo-controlled Phase IIb trial of the CFTR gene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers. Settings Data were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database. Participants Patients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV 1 ) between 50% and 90% predicted and any combination of CFTR mutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised). Interventions Subjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year. Main outcome measures The primary end point was the relative change in percentage predicted FEV 1 over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference. Results There was a significant ( p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) ( p = 0.031) and CT gas trapping ( p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age or CFTR mutation class. Subjects with a more severe baseline FEV 1 had a FEV 1 TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly ( p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups. Conclusions Monthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo. Limitations Although encouraging, the improvement in FEV 1 was modest and was not accompanied by detectable improvement in patients’ quality of life. Future work Future work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration. Trial registration ClinicalTrials.gov NCT01621867. Funding This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.