-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4417 Innovative Hematopoietic Gene-Therapy Concepts for Hereditary Pulmonary Alveolar Proteinosis Utilizing Hematopoietic Stem Cell Derived Macrophages

Gene Therapy and Transfer
Program: Oral and Poster Abstracts
Session: 801. Gene Therapy and Transfer: Poster III
Monday, December 7, 2015, 6:00 PM-8:00 PM
Hall A, Level 2 (Orange County Convention Center)

Nico Lachmann, PhD1,2,3*, Christine Happle, MD4*, Takuji Suzuki, MD5*, Miriam Hetzel1,2*, Kevin A. Link, PhD6, Adele Mucci1,2*, Paritha Arumugam, PhD7*, Jens Bankstahl, PhD8*, Dagmar Dilloo, MD9, Punam Malik, MD6*, Carolyn Lutzko, PhD6*, Axel Schambach, MD, PhD2*, Bruce Colston Trapnell, MD5*, Gesine Hansen, MD4* and Thomas Moritz, MD1,2*

1Reprogramming and Gene Therapy Group, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany, Hannover, Germany
2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
3JRG Translational Hematology of Congenital Diseases, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
4Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany
5Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH
6Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
7Translational Pulmonary Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
8Institute for Preclinical Molecular Imaging, Hannover Medical School, Hannover, Germany, Hannover, Germany
9Department of Pediatric Hematology and Oncology, Center for Child and Adolescent Medicine, Rheinische Friedrich-Wilhelms University, Bonn, Germany, Dusseldorf, Germany

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected  hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb-/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio.

Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages.

Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH