Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RA-Deficiency

Abstract

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP. To facilitate the translation of PMT therapy to human hPAP patients, a self-inactivating (SIN) lentiviral vector was generated expressing a codon-optimized human CSF2RA-cDNA driven from an EF1α short promoter (Lv.EFS.CSF2RA^coop), and a series of nonclinical efficacy and safety studies were performed in cultured macrophage cell lines and primary human cells. Studies in cytokine-dependent Ba/F3 cells demonstrated efficient transduction, vector-derived CD116 expression proportional to vector copy number, and GM-CSF-dependent cell survival and proliferation. Using a novel cell line constructed to express a normal GM-CSF receptor β subunit and a dysfunctional α subunit (due to a function-altering CSF2RA^G196R mutation) that reflects the macrophage disease phenotype of hPAP patients, it was demonstrated that Lv.EFS.CSF2RA^coop transduction restored GM-CSF receptor function. Further, Lv.EFS.CSF2RA^coop transduction of healthy primary CD34⁺ cells did not adversely affect cell proliferation or affect the cell differentiation program. Results demonstrate Lv.EFS.CSF2RA^coop reconstituted GM-CSF receptor α expression, restoring GM-CSF signaling in hPAP macrophages, and had no adverse effects in the intended target cells, thus supporting testing of PMT therapy of hPAP in humans.

Keywords: CSF2RA; HSCs; hPAP; hematopoietic gene therapy; lentivirus; macrophages.

Figure 1.

Vector design and functionality in the Ba/F3 cell line. (a) Schematic picture of the third-generation self-inactivating (SIN) lentiviral constructs Lv.EFS.CSF2RA^coop expressing the human codon-optimized cDNA of CSF2RA (upper picture) and Lv.EFS.GFP control vector (lower picture). (b) Human CD116 expression in untransduced (left) and Lv.EFS.CSF2RA^coop non-sorted transduced (right) Ba/F3 cells. (c) Human granulocyte/macrophage colony-stimulating factor (hGM-CSF)-dependent survival of Lv.EFS.GFP and Lv.EFS.CSF2RA^coop transduced and non-sorted Ba/F3 cells with mIL-3 as positive control, without cytokines as a negative control and increasing hGM-CSF concentrations (5–160 ng/mL; n = 3; two-way analysis of variance [ANOVA] using Sidak's post hoc testing). (d) Representative plots analyzing mCD131 and hCD116 expression in untransduced and Lv.EFS.CSF2RA^coop transduced Ba/F3 cells with defined vector copy numbers (VCNs) of 1, 2, and 4. (e) Mean fluorescence intensity (MFI) analysis of hCD116 expression in untransduced and Lv.EFS.CSF2RA^coop transduced Ba/F3 cells with defined VCNs of 1, 2, and 4 (left), and a bar graph depicting the CD116 MFI (right; n = 3; one-way ANOVA using Dunnett's post hoc testing). (f) hGM-CSF-dependent survival of Lv.EFS.CSF2RA^coop transduced Ba/F3 cells with defined VCNs of 1, 2, and 4 (n = 3; one-way ANOVA using Dunnett's post hoc testing). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Color images available online at www.liebertpub.com/hgtb

Figure 2.

Characteristics of the new murine alveolar macrophage cell line (mAM). (a) Morphology of the mAM parental line mAM (left) and mAM-hGM-R line expressing the human GM-CSFR (right); scale bar: ∼100 μm. (b) Representative histogram depicting hCD116 expression of mAM and mAM-hGM-R cells. (c) Western blot analysis of overall STAT5 expression and phosphorylated STAT5 in response to human and murine GM-CSF in mAM and mAM-hGMR cells to confirm species-specific response. No addition of cytokines (–) served as a negative control, human PBMCs as a positive control for hGM-CSF, the mouse cell line RAW264.7 as a positive control for mGM-CSF, and actin expression as loading control. (d) Western blot depicting dose-dependent phosphorylation of STAT5 in response to hGM-CSF (0–1,000 ng/mL) with actin expression as loading control. (e) Western blot depicting time-dependent phosphorylation of STAT5 after stimulation with hGM-CSF (0, 5, 15, 30, and 60 min of stimulation) with actin expression as loading control. PBMC, peripheral blood mononuclear cells; STAT5, signal transducer and activator of transcription 5; pSTAT5, phosphorylated STAT5. Color images available online at www.liebertpub.com/hgtb

Figure 3.

Vector analysis in the mAM cell line. (a) Morphology of the mAM-hPAP line expressing the mutated GM-CSFRα (mAM-hPAP); scale bar: ∼100 μm. (b) Representative histogram depicting hCD116 expression of mAM, mAM-hGM-R, and mAM-hPAP cells. (c) mAM-hPAP cells were transduced with Lv.EFS.CSF2RA^coop using multiplicities of infection (MOIs) of 0.1, 0.5, 1, and 10. Representative plots depicting hCD116 expression of mAM-hPAP and mAM-hPAP transduced with Lv.EFS.CSF2RA^coop. (d) MFI bargraph of hCD116 expression summarizing three independent transductions (n = 3; one-way ANOVA using Dunnett's post hoc testing). (e) Correlation between hCD116 MFI and VCN indicating a linear relationship between VCN and CD116 expression. (f) Southern blot analysis using a CSF2RA^coop probe to trace the transgene integrated to the genome of mAM-hPAP cells transduced with Lv.EFS.CSF2RA^coop. No transgene expression is detectable in mAM-hPAP cells, whereas the signal is increasing with higher MOIs (5, 10, 15, and 25) used to transduce the cells. Lentiviral transfer plasmid in different concentrations was used as a positive control. λ/H M = HindIII digested P32-labeled bacteriophage lambda DNA marker. (g) hGM-CSF uptake from cell culture medium. mAM and mAM-hPAP cells were not able to clear hGM-CSF from the cell culture supernatant, whereas mAM and mAM-hPAP^{Lv.EFS.CSF2RAcoop} cells efficiently cleared hGM-CSF over a period of 72 h. A well with no cells was used as a negative control. bp, base pairs. *p < 0.05; ****p < 0.0001. Color images available online at www.liebertpub.com/hgtb

Figure 4.

Transduction of primary CD34⁺ cells and macrophage differentiation. (a) Analysis of hCD116 expression in primary CD34⁺ cells, transduced with Lv.EFS.CSF2RA^coop (red), Lv.EFS.GFP (green), or mock-treated cells (black). Non-transduced and unstained CD34⁺ cells have been used as negative controls (gray). (b) Proliferation of transduced CD34⁺ cells, as determined by dilution of eFluor670. Mean fluorescent intensity values of e670 have been normalized to day 0 time point. (c) Total colony numbers per 1,500 input cells cultivated in methylcellulose containing human SCF, GM-CSF, IL-3, and Epo for 7–10 days. (d) Representative pictures of clonogenic cells observed after 7–10 days in methylcellulose-based assays. (e) Increase in overall cell number during differentiation of Lv.EFS.CSF2RA^coop (red), Lv.EFS.GFP (green), or mock treated cells (black) toward macrophages for 14 days in the presence of IL-3, IL-6, FLT3, M-CSF, and GM-CSF. (f) Representative bright-field images of macrophages on day 14 of differentiation. (g) Phenotypic analysis of macrophages on day 14 of differentiation by flow cytometry. Histograms are shown as overlays of mock-treated (blue), Lv.EFS.CSF2RA^coop (black), and Lv.EFS.GFP (green) cells. (h) Functional analysis of transduced macrophages in the presence of hGM-CSF. Histograms show phosphorylation of STAT5 in the absence (gray) or presence (black) of hGM-CSF. (i) Relative amount of pSTAT5 after stimulation of transduced macrophages with hGM-CSF. Values are calculated and normalized to non-stimulated conditions. Color images available online at www.liebertpub.com/hgtb