Page 306 - Haematologica April 2020

M. Hetzel et al.
and IL-5 receptor. Although the GM-CSFR is expressed on a wide variety of hematopoietic cells, mutations in either CSF2RA2,3 or CSF2RB4,5 almost exclusively affect the maturation and functionality of AM, leading to dis- turbed intracellular surfactant degradation, intrapul- monary deposition of phospholipids and lipoproteins, and, in turn, severe respiratory insufficiency. Current treatment options are symptomatic only and consist of vigorous treatment of pulmonary infections and whole lung lavage, a highly invasive procedure performed under general anesthesia, which may need to be repeated every 4-8 weeks. As for other hereditary diseases affecting the lymphohematopoietic system, allogeneic hematopoietic stem cell transplantation (alloHSCT) or hematopoietic stem cell-based gene therapy (HSC-GT) using retroviral vectors6,7 potentially offer a permanent cure for herPAP patients and proof of concept for these concepts has been established in murine models.8,9 However, in the clinical setting, both procedures are problematic due to the required chemo/radiotherapeutic preconditioning and the poor pulmonary status of herPAP patients.3 On the other hand, over the last decade, substantial progress has been made in the fields of alloHSCT as well as HSC-GT, and in this context, successful alloHSCT has recently been reported in two patients suffering from secondary PAP10 as well as in a herPAP patient.11
In addition, novel treatment strategies for herPAP are evolving. Thus, the direct intrapulmonary application of macrophages (pulmonary macrophage transplantation; PMT) has demonstrated profound and long-lasting thera- peutic efficacy in two murine models of herPAP in the absence of any preconditioning.12-16 While it is not clear yet whether in the human setting these pulmonary administered macrophages will permanently persist in the lungs and provide a long-term cure for herPAP patients, PMT might still improve the patients’ general condition to allow for a more permanent approach such as alloHSCT or HSC-GT.
On this background, we here employed a state-of-the- art, 3rd generation self-inactivating (SIN) lentiviral vector and transduced lineage-negative hematopoietic stem and progenitor cells (lin– HSPC) derived from the bone mar- row (BM) of Csf2rb-/- mice17 to evaluate the feasibility and efficacy of an HSC-GT strategy for herPAP in vivo. In this study, we demonstrated robust and long-term pulmonary engraftment of AM which was associated with signifi- cant improvements of critical disease-related parameters such as elevated protein, cholesterol, or cytokine levels in the alveolar spaces. In addition, our study offers novel insights into the early reconstitution phase of a functional AM compartment following HSCT.
Methods
Mice
B6.SJL-Ptprca-Pep3b/BoyJZtm (Ly5.1; CD45.1) mice were used as wild-type (WT) controls, and B6;129P2- Csf2rb2tm1Mur (Csf2rb-/-) harboring a knockout in the Csf2rb gene served as a disease model of PAP. Mice were housed in the central animal facility of Hannover Medical School under specific-pathogen-free conditions in individually ventilated cages (IVC). Mice had access to food and water ad libitum. All animal experiments were approved by the Lower Saxony State animal welfare committee
and were conducted according to the law governing ani- mal welfare.
Isolation, cultivation, and transduction of lineage negative cells
Lineage negative cells were isolated from total bone marrow using lineage cell depletion kit (Miltenyi) following the manufac- turer’s instructions. Pre-stimulation of lineage negative cells was performed for 24 hours in STIF medium (StemSpan medium sup- plemented with 1mM Penicillin/Streptomycin, 2mM L-glutamine, 100 ng/mL mSCF, 20 ng/mL TPO, 20 ng/mL IGF-2 and 100 ng/mL FGF2). Following this, transduction of lineage negative cells was performed on RetroNectin® (Takara) coated plates using STIF medium and MOI between 10 and 20, as previously described.18 For in vitro assays, cells were sorted for GFP expression using a FACSAria IIu.
Hematopoietic stem cell transplantation
Age-matched (12-16 week old) Csf2rb-/- recipient mice of both sexes were irradiated with a single dose of 9 Gy. Lin– BM cells of wild-type (WT) donor mice were isolated as described above and injected intravenously into the tail vein of recipient mice 24 hours post irradiation. Alternatively, lineage negative cells of Csf2rb-/- donor mice were isolated and transduced with SIN lentiviral gene therapy or GFP control vectors and transplanted by the same pro- tocol. In secondary transplantation, BM of primary recipients was isolated, red blood cell lysis performed and subsequently, cells were injected by the same protocol as described above. For all experiments, non-sex- and non-age-matched (age ≤9 months), non-irradiated Csf2rb-/- and WT mice served as reference controls.
Bronchoalveolar lavage
After closing the left lung, the right lung was flushed three times with PBS (once with 0.6 mL and 2 times with 0.5 mL) to obtain bronchoalveolar lavage fluid (BALF). The turbidity of the fluid was measured by analyzing the OD600 value on a BioPhotometer (Eppendorf). The protein concentration of total BALF was ana- lyzed using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. BALF sam- ples were pelleted for 10 minutes at 400xg. Supernatants were used to determine the concentration of GM-CSF, M-CSF, and MCP-1 by ELISA (Mouse Quantikine Kits, R&D Systems), as described.2 Cholesterol levels were measured by the amplex red cholesterol assay (Life Technologies) according to the manufactur- er’s protocol, as described.19 Pelleted cells were stained and ana- lyzed by flow cytometry using BD LSR II.
Statistical analysis
Statistical analysis was performed using the Prism 7 software (GraphPad) applying ANOVA and recommended post hoc testing. Unless otherwise stated, bars and average numbers in the text rep- resent mean±standard error of mean (SEM).
Results
Lentiviral transduction restores GM-CSFRβ expression and GM-CSF-dependent functionality in Csf2rb-/- hematopoietic stem and progenitor cells and macrophages in vitro
A 3rd generation SIN lentiviral vector constitutively expressing murine Csf2rb cDNA from an elongation factor 1 α (EF1 α) promoter (Lv.Csf2rb) was employed, and for better visualization upon flow cytometry, an enhanced GFP (eGFP) reporter coupled to the transgene via an inter-
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