Page 308 - Haematologica April 2020

M. Hetzel et al.
cells were observed when a cocktail of IL-3, IL-6, SCF, and EPO was applied (Online Supplementary Figure S1D and E) demonstrating the GM-CSF specificity of Lv.Csf2rb-medi- ated effects.
Given that primarily macrophages (MΦ) are affected in herPAP, as a next step, lin– cells were differentiated towards MΦ in vitro by applying M-CSF and assessed for morphology, transgene expression, and GM-CSF-depen- dent functionality. Typical MΦ morphology similar to WT and Csf2rb-/- MΦ was observed for Lv.Csf2rb-transduced MΦ (Online Supplementary Figure S1F) and sustained GFP as well as CD131 expression was detected for these cells (Figure 1D and E). Moreover, Lv.Csf2rb-transduced MΦ, in clear contrast to non-transduced Csf2rb-/- MΦ, were able to bind and internalize GM-CSF with comparable efficiency as WT MΦ (Figure 1F). In addition, when we analyzed phosphorylation of the signal transducer and activator of transcription 5 (STAT5), a major component in the GM- CSFR signaling cascade, non-transduced Csf2rb-/- MΦ failed to phosphorylate STAT5 in response to GM-CSF, whereas STAT5 phosphorylation was restored following Lv.Csf2rb-transduction (Figure 1G).
A
These data demonstrate that the Lv.Csf2rb vector effi- ciently directs Csf2rb expression and restores GM-CSF- dependent functionality in Csf2rb-/- HSPC as well as there- of derived MΦ, whereas GM-CSF independent properties such as colony formation potential or differentiation prop- erties in response to other cytokines were not affected. Thus, this vector appeared suitable for further evaluation in vivo.
Lv.Csf2rb-transduced lin– hematopoietic stem and progenitor cells engraft in Csf2rb-/- mice and reconstitute the hematopoietic system
For in vivo evaluation of our construct, we performed HSC-GT employing lethally irradiated Csf2rb-/- mice and either the Lv.Csf2rb construct or a control construct harbor- ing GFP only (Lv.GFP) (Figure 2A). A cohort receiving healthy CD45.1+ donor cells served as a positive control. To monitor engraftment and hematopoietic reconstitution, blood samples were taken four, eight, and twelve weeks after HSC-GT, the latter representing the time point of final analysis. Two independent rounds of transplantation were performed with a total of six mice receiving Lv.Csf2rb-
BCD
Figure 2. Hematopoietic reconstitution after Lv.Csf2rb hematopoietic stem cell-based gene therapy (HSC-GT). (A) Schematic representation of HSC-GT procedure. Csf2rb-/- hematopoietic stem and progenitor cells (HSPC) were isolated from donor mice and transduced with the gene therapy vector (Lv.Csf2rb) or a GFP control vector (Lv.GFP). Lv.Csf2rb-, Lv.GFP-transduced or healthy CD45.1 control HSPC were injected into Csf2rb-/- recipients previously lethally irradiated with a single dose of 9 Gy. Blood samples were drawn four, eight and 12 weeks after HSC-GT. The final analysis was performed 12 weeks after HSC-GT to evaluate engraftment of donor cells in bone marrow (BM), blood and lungs as well as to evaluate the reduction of lipoproteinaceous material in the lungs. All images in this illustration were adapted from Servier Medical Art (https://smart.servier.com) and are distributed under the CC-BY 3.0 license. (B) Blood count analysis showing total number of white blood cells (WBC) per mm3 of peripheral blood four, eight and 12 weeks after HSC-GT in mice receiving Lv.Csf2rb-transduced Csf2rb-/- cells (black) or healthy CD45.1+ cells (gray); n=6 in two independent experiments. Dots indicate mean±standard error of mean. (C) Percentage of GFP+ cells in peripheral blood CD45+ cells four, eight and 12 weeks after injection of Lv.Csf2rb-transduced cells; n=6 in two independent experiments. Circles belong to the first experiment, squares to the second experiment; equal symbols in (C) and (D) represent the same mice. (D) Percentage of GFP+ cells in peripheral blood CD45+, CD19+, CD3+ and Gr-1+ subsets of mice transplanted with Lv.Csf2rb-transduced cells; n=6 in two independent experiments.
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