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HSC-GT for herPAP
transduced cells, three mice receiving Lv.GFP-transduced cells, and four mice receiving healthy CD45.1+ cells. Hematopoietic reconstitution in mice receiving Lv.Csf2rb- transduced cells was detected already at four weeks (6.0±0.9x103/mm3 total white blood cells; WBC; mean±SEM), and WBC maintained stably thereafter until week 12 comparable to control mice transplanted with healthy CD45.1+ HSPC (Figure 2B). A clear, though low- level, contribution of Lv.Csf2rb-transduced GFP+ cells was observed during this period (Figure 2C) with a comparable contribution to T, B, and myeloid compartments when ana- lyzed at the week 12 time point (Figure 2D).
Thus, all mice transplanted with Lv.Csf2rb-transduced cells presented effective reconstitution of the hematopoi- etic system, although at low levels of transduced cells.
Lv.Csf2rb-transduced cells migrate to the lungs and restore the alveolar macrophage pool
In a next step, we evaluated the engraftment of gene- modified cells in the alveolar spaces of Csf2rb-/- recipients following HSC-GT. While at the end of the experiment both Csf2rb-/- mice and control mice transplanted with Lv.GFP-transduced cells lacked endogenous AM, mice transplanted with Lv.Csf2rb-transduced cells similar to WT mice and controls transplanted with healthy CD45.1+ cells presented a distinct population of Siglec-F+ cells in their lungs, which resided in the alveolar spaces close to the alveolar septa (Online Supplementary Figure S2A). To analyze these cells in more detail, homogenized lung tis- sue was analyzed by flow cytometry (Figure 3A). Cells displaying a CD45+F4/80+Siglec-F+CD11chigh phenotype characteristic of mature AM were detected in all mice transplanted with Lv.Csf2rb-transduced cells, similar to control animals receiving healthy CD45.1+ cells or WT mice, while these cells were absent in Csf2rb-/- controls or mice transplanted with Lv.GFP-transduced cells (Figure 3B and Online Supplementary Figure S2B).
Corresponding results were obtained when the lungs were flushed, and the resulting bronchoalveolar lavage fluid (BALF) was analyzed by flow cytometry. As expect- ed, AM were detected in mice transplanted with Lv.Csf2rb-transduced or healthy CD45.1+ HSPC and in WT mice (Figure 3C and D and Online Supplementary Figure S2C). The majority of hematopoietic cells in the BALF of mice transplanted with Lv.Csf2rb-transduced cells was GFP+ and showed robust CD131 expression comparable to mice transplanted with healthy CD45.1+ cells. In contrast, GFP+ cells were almost absent in mice receiving Lv.GFP-transduced cells (Figure 3E and Online Supplementary Figure S2D). Vector copy number (VCN) analysis of BALF cells confirmed the presence of geneti- cally modified cells in the lungs of mice receiving Lv.Csf2rb- or Lv.GFP-transduced cells. However, mice receiving Lv.GFP-transduced cells exhibited similar VCN in BM, peripheral blood (PB), or BALF cells (Online Supplementary Figure S2E), whereas mice of the Lv.Csf2rb group showed substantially increased VCN in BALF as compared to BM or PB cells (Figure 3F). Enrichment of VCN in BALF versus BM cells was up to 16-fold, docu- menting the enhanced recruitment of corrected cells to the alveolar spaces in the Lv.Csf2rb-transduced cohort.
Taken together, these data demonstrate that, following HSC-GT, in the Lv.Csf2rb group a population of trans- duced cells migrates to the alveolar spaces and restores the deficient AM pool of Csf2rb-/- recipients.
Lv.Csf2rb hematopoietic stem cell-based gene therapy reverses the hereditary pulmonary alveolar proteinosis disease phenotype
A major hallmark of herPAP is the accumulation of sur- factant proteins and lipids in the alveolar spaces giving rise to a turbid, milky appearance of the BALF. Following the transplantation of Lv.Csf2rb-transduced HSPC, however, the BALF acquires a clear appearance comparable to WT mice or mice transplanted with healthy CD45.1+ cells (Figure 4A). This initial observation was confirmed by BALF turbidity (OD600 absorbance) and total protein analysis (Figure 4B and C). As the material accumulated in the alveolar spaces is characterized by a high contribution of cholesterol,19 we also analyzed the cholesterol levels in BALF and detected significant improvements in mice transplanted with Lv.Csf2rb-transduced cells (Figure 4D). Moreover, in Csf2rb-/- mice, like in herPAP patients, BALF levels of GM-CSF, M-CSF and MCP-1 are markedly increased due to auto-regulatory mechanisms directed to stabilize the AM population.1-5,21 While none of these parameters was improved by transplantation of Lv.GFP- transduced cells, twelve weeks after injection of Lv.Csf2rb-transduced cells, all parameters normalized mimicking the situation in WT or CD45.1 transplanted control animals (Figure 4E-G), highlighting the consider- able potential of HSC-GT in herPAP.
As a next step, we evaluated lung sections for the depo- sition of Periodic-Acid Schiff (PAS) positive material. While lungs of untreated Csf2rb-/- mice and animals trans- planted with Lv.GFP-transduced cells nicely showed the pathognomonic foci of accumulated PAS+ surfactant mate- rial, these foci were virtually absent in mice transplanted with Lv.Csf2rb-transduced cells similar to animals of the CD45.1 and WT cohort (Online Supplementary Figure S3A). Morphometric quantification confirmed the pronounced reduction of PAS+ areas following transplantation of Lv.Csf2rb-transduced cells (Figure 4H).
Given the characteristic ground-glass opacities in the lungs of herPAP patients on computed tomography (CT) scans, we also performed CT scans in our mice twelve weeks after HSC-GT and quantified lung densities during the inspiratory and expiratory phases (Figure 4I-K). In line with expectations and our data from BALF analysis, CT scans confirmed significant improvements in inspiration and expiration lung densities following Lv.Csf2rb HSC- GT when compared to non-treated Csf2rb-/- mice or the Lv.GFP cohort. These radiographic data were corroborat- ed by mechanical data of the lung demonstrating improved static compliance and inspiratory capacity in Csf2rb-/-mice upon transplantation of Lv.Csf2rb-trans- duced cells (Online Supplementary Figure S3B and C).
In summary, these data demonstrate that HSC-GT employing the Lv.Csf2rb vector significantly improves and (concerning some of these parameters) even com- pletely normalizes the herPAP disease phenotype.
Lv.Csf2rb hematopoietic stem cell-based gene therapy provides long-term cure for hereditary pulmonary alveolar proteinosis disease phenotype
To better assess the long-term potential of our HSC-GT approach, we performed serial transplantation to second- ary recipients. Indeed, in half of the secondary recipients, robust pulmonary engraftment of AM associated with pronounced clinical benefit similar to primary recipients was observed indicated by decreased turbidity and
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