1. Volume 16, Issue 4, Pages 757-764 (April 2008)
Gene Therapy of βc-Deficient Pulmonary Alveolar Proteinosis (βc-PAP): Studies in a Murine in vivo Model 
Veronika Kleff, Ursula R Sorg, Carsten Bury, Takuji Suzuki, Ina Rattmann, Moran Jerabek-Willemsen, Christopher Poremba, Michael Flasshove, Bertram Opalka, Bruce Trapnell, Uta Dirksen, Thomas Moritz 
Molecular Therapy 
Volume 16, Issue 4, Pages (April 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Retroviral vectors and experimental design. (a) Hybrid spleen focus forming virus/murine embryonic stem cell virus–based vectors containing the complementary DNA (cDNA) for the murine common β-chain (mβc) of the interleukin-3 (IL-3)/IL-5/granulocyte–macrophage colony-stimulating factor receptor or for the enhanced green fluorescent protein (EGFP) in combination with the cDNA coding for the O 6 -methylguanine-DNA-methyltransferase mutant MGMTP140K. (b) RhIL-6/rmSCF prestimulated bone marrow (BM) cells from βc–/– or C57Bl/6 wild-type mice (WT) were transduced with the SF91-mβc-IRES-MGMTP140K or the SF91-EGFP-IRES-MGMTP140K vectors. Subsequently, cells were either seeded in colony forming unit (CFU) assays or transplanted into lethally irradiated βc–/– mice. Upon hematopoietic reconstitution, a cohort of animals was treated with combined O6-benzylguanine (BG)/temozolomide (TMZ) chemotherapy (CTX). FN, fibronectin; IRES, internal ribosomal entry site; LTR, long-terminal repeat; mβc–/–, murine β-chain-deficient; rmSCF, recombinant murine stem cell factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
SF91-mβc-IRES-MGMTP140K transduction induces chemotherapy (CTX) resistance and restores granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent clonogenic growth and GM-CSF sensitivity in primary mβc–/– clonogenic progenitor cells. (a and b) Colony numbers (mean ± SD) recovered from methylcellulose cultures supported by recombinant murine GM-CSF (rmGM-CSF) alone (n = 11) or recombinant human G-CSF, recombinant human erythropoietin, recombinant murine stem cell factor (n = 6) are given for SF91-mβc-IRES-MGMTP140K-transduced (white column) and nontransduced control (black column) mβc–/– bone marrow (BM) cells. (c–e) Clonogenic growth of SF91-mβc-IRES-MGMTP140K-transduced mβc–/– BM cells as well as nontransduced and SF91-mβc-IRES-MGMTP140K-transduced C57Bl/6 wild-type BM cells in the presence of increasing concentrations of rmGM-CSF (0–1,000 ng/ml) is depicted. *Denotes significant differences (P < 0.001) by Mann–Whitney U test. CFU-C, colony forming unit cells; IRES, internal ribosomal entry site; mβc–/–, murine β-chain-deficient.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
mβc Gene transfer in a murine in vivo model: effect on hematopoiesis. Bone marrow (BM)-derived colonies grown in the presence of recombinant murine interleukin-3 (rmIL-3) (gray bars) or recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (black bars) at the end of the experiment are depicted. One representative experiment is shown. (a) Colonies derived from unmanipulated wild-type (WT) mice, chemotherapy-treated (CTX+) and nontreated (CTX–) βc–/– mice transplanted with SF91-EGFP-IRES-MGMTP140K-transduced wild-type cells (WT* → mβc–/–), unmanipulated βc–/– mice (mβc–/–), and chemotherapy-treated (CTX+) and nontreated (CTX–) βc–/– mice receiving transduced mβc–/– cells (mβc–/–* → mβc–/–) are depicted. (b) Enhanced green fluorescent protein (EGFP) transgene expression from BM cells of chemotherapy-treated (CTX+) and nontreated (CTX–) βc–/– mice transplanted with SF91-EGFP-IRES-MGMTP140K-transduced mβc–/– cells (mean ± SD, n = 3). (c) Colony numbers derived from βc–/– mice receiving genetically corrected mβc–/– cells with (CTX+) and without (CTX–) chemotherapy treatment are given. The transduction rate was estimated based on the colony formation in the presence of rmIL-3 or rmGM-CSF as follows: the ratio of rmGM-CSF- to rmIL-3-responsive colonies of normal WT cells (see a) as well as of SF91-mβc-IRES-MGMTP140K-transduced βc–/– cells following chemotherapy, where ≥ 80% transgene expression is expected, was ∼1:2. Therefore, the 10 rmGM-CSF-responsive colonies derived from transduced cells in animals not treated by chemotherapy would correspond to five rmIL-3-responsive colonies (dotted line), representing ∼13.5% (5/37) of total rmIL-3-responsive colonies. *Denotes cells transduced with SF91-EGFP-IRES-MGMTP140K or SF91-mβc-IRES-MGMTP140K as indicated. CFU-C, colony forming unit cells; IRES, internal ribosomal entry site; mβc–/–, murine β-chain-deficient.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Reversal of lung pathology following transplantation of mβc–/–-transduced bone marrow cells. Lung tissue sections stained with periodic acid Schiff (PAS) are shown (scale bar = 50 μm). (a) Normal lung tissue from a C57Bl/6 wild-type mouse. (b and c) Pathologic lung tissue of an unmanipulated βc–/– mouse is characterized by accumulation of PAS-positive material in the alveolar spaces and focal peribronchiovascular lymphoid infiltrates (*). (d) Pathological lung tissue of a βc–/– control mouse receiving SF91-EGFP-IRES-MGMTP140K-transduced BM cells. (e–g) Lung tissues of βc–/– mice receiving SF91-mβc-IRES-MGMTP140K-transduced mβc–/– BM cells are depicted. PAS-positive material in the alveolar spaces has disappeared except for occasional small areas (g) Irrespective of treatment with O6-benzylguanine/temozolomide or not. BM, bone marrow; EGFP, enhanced green fluorescent protein; IRES, internal ribosomal entry site; mβc–/–, murine β -chain-deficient.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
mβc Protein levels following SF91-mβc-IRES-MGMTP140K transduction. Western blot analysis shows mβc-protein levels from spleen cells of unmanipulated βc–/– and C57Bl/6 mice as well as βc–/– mice transplanted with SF91-EGFP-IRES-MGMTP140K- or SF91-mβc-IRES-MGMTP140K-transduced mβc–/– BM cells. Mice were either chemotherapy-treated (CTX+), or not (CTX–). mβc Blots were exposed for 15 or 180 seconds. EGFP, enhanced green fluorescent protein; IRES, internal ribosomal entry site; mβc–/–, murine β-chain-deficient; WT, wild type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions