Gene modified macrophages as therapies for respiratory diseases
File(s)
Author(s)
Clarke, Nora
Type
Thesis
Abstract
Gene therapy in the context of pulmonary disease often targets gene transfer to the lung epithelium. Recently there has been a growing interest in alternative macrophage-based cell therapies, where following ex vivo modifications, macrophages are transplanted to the lung to target inherent macrophage dysfunction or drive expression of a secreted therapeutic protein in vivo.
Here I demonstrated successful ex vivo transduction of bone marrow-derived macrophages (BMDMs) by a proprietary fusion (F) and haemagglutinin (HN) pseudotyped simian immune deficiency (SIV) lentiviral vector (rSIV.F/HN) encoding a chimeric hybrid cytomegalovirus enhancer/elongation factor 1 alpha (hCEF) promoter. Following pulmonary transplantation of these modified macrophages, production of secreted transgenic proteins was detected in the lungs, where in wildtype mice, in vivo pulmonary expression of a Gaussia luciferase secreted reporter was sustained for up to 16 weeks in bronchoalveolar lavage fluid.
In autoimmune pulmonary alveolar proteinosis (aPAP) anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies lead to defective surfactant clearance and deposition in alveoli. Ongoing clinical trials suggest the benefit of therapeutic interventions aiming to increase bioavailability of GM-CSF with recombinant protein therapy. Proof-of-concept for this platform macrophage-based technology was therefore performed in a mouse model of aPAP (GM-CSF knockouts) delivering macrophages transduced with a GM-CSF transgene, where four-weeks post-transplantation biomarkers of aPAP disease improved in GM-CSF treated mice in comparison to Glux controls.
To increase the efficacy of this platform technology, I also investigated gene transfer optimisation strategies. Here, the F/HN pseudotype reported similar transduction efficiencies to the gold-standard vesicular stomatitis virus G glycoprotein (VSV-G) pseudotype. Promoter comparisons between hCEF, elongation factor 1 alpha promoter (EF1a) and a cytomegalovirus enhancer/promoter (CMV), demonstrated that following transduction, more macrophages are positive for transgene expression using an EF1a promoter, while the relative strength of CMV was significantly higher than hCEF and EF1α. Additionally, transducing monocyte precursors during macrophage differentiation significantly increased transduction efficiency across multiplicities of infection.
In cystic fibrosis, patients have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and research suggests that intrinsic loss-of-function mutations in alveolar macrophages reduce anti-microbial efficacy and promote pro-inflammatory phenotypes. To determine if this platform technology could target these defects, I attempted to characterize the bactericidal activity of macrophages lacking functional CFTR in vitro. However, no difference in bacterial phagocytosis or killing of internalized bacteria was observed between BMDMs derived from wildtype and CFTR knockout mice.
This work presents initial work towards the development of an ex vivo macrophage-based platform technology for pulmonary disease. I have demonstrated proof-of-concept for pre-clinical efficacy of secreted transgene expression in a model of aPAP and lay the foundation for the treatment of diseases caused by alveolar macrophage dysfunction or a lack of secreted proteins like aPAP and alpha-1 antitrypsin deficiency.
Here I demonstrated successful ex vivo transduction of bone marrow-derived macrophages (BMDMs) by a proprietary fusion (F) and haemagglutinin (HN) pseudotyped simian immune deficiency (SIV) lentiviral vector (rSIV.F/HN) encoding a chimeric hybrid cytomegalovirus enhancer/elongation factor 1 alpha (hCEF) promoter. Following pulmonary transplantation of these modified macrophages, production of secreted transgenic proteins was detected in the lungs, where in wildtype mice, in vivo pulmonary expression of a Gaussia luciferase secreted reporter was sustained for up to 16 weeks in bronchoalveolar lavage fluid.
In autoimmune pulmonary alveolar proteinosis (aPAP) anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies lead to defective surfactant clearance and deposition in alveoli. Ongoing clinical trials suggest the benefit of therapeutic interventions aiming to increase bioavailability of GM-CSF with recombinant protein therapy. Proof-of-concept for this platform macrophage-based technology was therefore performed in a mouse model of aPAP (GM-CSF knockouts) delivering macrophages transduced with a GM-CSF transgene, where four-weeks post-transplantation biomarkers of aPAP disease improved in GM-CSF treated mice in comparison to Glux controls.
To increase the efficacy of this platform technology, I also investigated gene transfer optimisation strategies. Here, the F/HN pseudotype reported similar transduction efficiencies to the gold-standard vesicular stomatitis virus G glycoprotein (VSV-G) pseudotype. Promoter comparisons between hCEF, elongation factor 1 alpha promoter (EF1a) and a cytomegalovirus enhancer/promoter (CMV), demonstrated that following transduction, more macrophages are positive for transgene expression using an EF1a promoter, while the relative strength of CMV was significantly higher than hCEF and EF1α. Additionally, transducing monocyte precursors during macrophage differentiation significantly increased transduction efficiency across multiplicities of infection.
In cystic fibrosis, patients have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and research suggests that intrinsic loss-of-function mutations in alveolar macrophages reduce anti-microbial efficacy and promote pro-inflammatory phenotypes. To determine if this platform technology could target these defects, I attempted to characterize the bactericidal activity of macrophages lacking functional CFTR in vitro. However, no difference in bacterial phagocytosis or killing of internalized bacteria was observed between BMDMs derived from wildtype and CFTR knockout mice.
This work presents initial work towards the development of an ex vivo macrophage-based platform technology for pulmonary disease. I have demonstrated proof-of-concept for pre-clinical efficacy of secreted transgene expression in a model of aPAP and lay the foundation for the treatment of diseases caused by alveolar macrophage dysfunction or a lack of secreted proteins like aPAP and alpha-1 antitrypsin deficiency.
Version
Open Access
Date Issued
2021-10
Date Awarded
2022-03
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Griesenbach, Uta
Alton, Eric
Grant Number
WHRR-I34006
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)