CRISPR/Cas9 genome editing therapy of hereditary pulmonary alveolar proteinosis

In the Best of Paediatrics session, Dr Kenjiro Shima (Cincinnati Children’s Hospital, USA) provided proof-of-principle ex vivo data that CRISPR/Cas9-mediated genome editing corrects macrophage function in hereditary pulmonary alveolar proteinosis patient cells [1].

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disease characterised by surfactant accumulation in pulmonary alveoli resulting in respiratory failure. The disease is caused by mutations in the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha or beta subunit genes (CSF2RA or CSF2RB) resulting in impaired GM-CSF-dependent surfactant clearance by alveolar macrophages. Typical CT and histological hallmarks of the disease are used for diagnosis. Dr Shima’s team has previously generated human induced pluripotent stem cells (iPSCs) from hPAP patients [2]. iPSCs are derived from the somatic cells (in this case, fibroblasts from a skin biopsy) and reprogrammed back into a pluripotent state. The pluripotent iPSCs can then be differentiated to a wide range of tissues and cell types. iPSCs can be used for in vitro disease modelling, in vitro drug screening, and patient-specific cell therapy investigations. In rare diseases like hPAP, it can be difficult to acquire patient samples, thus iPSC cells are a useful tool to investigate pathogenesis. Dr Shima and colleagues characterised iPSC-derived macrophages that retained disease-specific features in functional tests of GM-CSF signalling. The authors had previously demonstrated that lentiviral vector-mediated reconstitution of CSF2RA restored GM-CSF signalling and function in hPAP iPSC-derived macrophages derived from a patient with CSF2RA mutations [2]. The team hypothesised that CRISPR/Cas9 genome editing could repair the impaired function of hPAP-iPSC-derived macrophages and overcome the limitations of viral vector-mediated gene therapy such as insertional mutagenesis and pose a better potential therapy for patients.

The researchers generated wild-type CSF2RA guide-RNA for CRISPR/Cas9 genome editing aimed at correcting the truncating mutation CSF2RA^R217X (CSF2RAc.649C>T gene mutation). The double stranded break generated by CRISPR/Cas9 was repaired by homology-directed repair using the provided wild-type guide RNA, to correct the gene mutation in situ.

The investigators then terminally differentiated the “corrected” patient fibroblasts to macrophages, in two steps: first by using a kit to differentiate to haematopoietic stem cells, and then to further differentiate to macrophages by culturing in the presence of GM-CSF and macrophage colony-stimulating factor, and performed functional analyses compared with the original (isogenic) patient fibroblasts. Firstly, macrophage surface markers were confirmed in all clones used for functional assays (i.e. CD14, CD115, CD49d, CD68, CD11b, CD163, HLA-DR). Of note, the makers CD116 and CD206, involved in GM-CSF signalling, were impaired in the original patient fibroblasts, but were rescued in the “corrected” patient-derived macrophages. The researchers then evaluated GM-CSF signalling by GM-CSF clearance assay. The gene-corrected macrophages cleared the GM-CSF in a comparable manner to wild-type cells, while no clearance was observed in the uncorrected original patient-derived differentiated macrophages. Downstream of GM-CSF signalling is STAT5, which becomes phosphorylated when activated. While the patient-uncorrected macrophages did not demonstrate any STAT5 phosphorylation due to the gene mutation, the gene-corrected macrophages showed a fully restored response. Likewise, PPARG (peroxisome proliferator-activated receptor gamma) gene expression is essential for alveolar macrophages, and it is downregulated in hPAP macrophages, but Dr Shima showed that it was entirely restored in gene-corrected macrophages. Cell proliferation upon stimulation of GM-CSF was also restored in gene-corrected macrophages. Proinflammatory signalling, as measured by tumour necrosis factor alpha release after exposure to lipopolysaccharide, indicated that the impaired activation of this response in the patient macrophages was fully restored, as was cholesterol clearance function using fluorescent cholesterol.

In conclusion, CRISPR/Cas9 genome editing successfully repaired the function of GM-CSF receptor in hPAP iPSCs. These results suggest that genome editing therapy is a promising strategy to further develop for treating hPAP patients in the future.

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   1. Shima K, et al. A4004, ATS 2019, 17-22 May, Dallas, USA.
   2. Suzuki T, et al. Am J Respir Crit Care Med. 2014 Jan 15;189(2):183-93.

Posted on 23 July, 201925 March, 2024