783. Correction of βc-Deficient Pulmonary Alveolar Proteinosis (PAP) by Hematopoietic Stem Cell Gene Transfer in a Murine Model
Authors: V. Kleff, U.R. Sorg, C. Bury, B. Opalka, M. Flasshove, U. Dirksen, T. Moritz
Journal: Molecular Therapy (2006)

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PAP due to deficiency of the common signal transduction chain (|[beta]|c) of the L3/IL5/GM-CSF receptor superfamily is a rare and rapidly fatal monogeneic disease with functional insufficiency of with the pulmonary macrophages. While hematopoietic stem cell gene therapy represents a potentially curative therapy the defect is not functionally manifested in stem cells and stable enrichment of genetically corrected cells in vivo will require an additional selection system.

Thus, we have generated a retroviral vector construct (SF91- m|[beta]|c-IRES-MGMTP140K) expressing the murine |[beta]|c (m|[beta]|c) cDNA in combination with the in vivo selectable drug resistance gene MGMTP140K coding for an O6-benzylguanine (BG) resistant point mutation of the DNA-repair-protein O6-methylguanine-DNA- methyltransferase and utilized this construct to transduce hematopoietic progenitor and stem cells in a murine model of m|[beta]|c- deficient (m|[beta]|c-/-) PAP.

Our construct functionally restores m|[beta]|c activity as demonstrated by significantly enhanced colony formation in the presence of GM- CSF upon m|[beta]|c transduction of m|[beta]|c-/- bone marrow cells (1|[plusmn]|0.5 vs. 9|[plusmn]|5 CFU-C/1x105, p=0.002; n=11). Control cultures set up with G-CSF, EPO and SCF indicated the GM-CSF specificity of the restored cytokine sensitivity. The GM-CSF sensitivity profile of |[ldquo]|repaired|[rdquo]| m|[beta]|c-/- cells matched that of non-transduced but also of m|[beta]|c-transduced wildtype bone marrow cells. Functional expression of MGMTP140K was demonstrated by increased resistance of progenitor cell-derived colonies to 10|[mu]|M BG plus 25 to 200 |[mu]|g/ml TMZ following SF91-m|[beta]|c-IRES-MGMTP140K transduction. Significant in vitro enrichment of genetically corrected m|[beta]|c-/- cells was shown when SF91-m|[beta]|c-IRES-MGMTP140K transduced cells were exposed to 10|[mu]|M BG plus 0, 50 or 100 |[mu]|g/ml TMZ before clonogenic culture (11.4|[plusmn]|4.3% (0 |[mu]|g/ml TMZ) versus 36.4|[plusmn]|9.6% (50 |[mu]|g/ml TMZ, p=0.034) or 73.7|[plusmn]|19.2% (100 |[mu]|g/ml TMZ, p=0.015) (n=6)).

To analyse the effect of m|[beta]|c gene transfer in our in vivo PAP model, SF91-m|[szlig]|c-IRES-MGMTP140K transduced |[szlig]|c-/- cells were transplanted into lethally irradiated |[szlig]|c-/- recipient mice which upon hematopoietic recovery were treated with BG/TMZ for 5 weeks. When animals were sacrificed at the end of the experiment, dramatic improvements in lung pathology were noted. Lung sections showed reduction of PAS positive regions in gene corrected animals to near normal levels and, furthermore, a radical reduction of lymphoid infiltration. GM-CSF-dependent colony function was restored by bone marrow cells and functional as well as flow cytometric analysis demonstrated substantial enrichment of genetically corrected cells in the bone marrow by BG/TMZ application (from 10|[ndash]|20% to 50|[ndash]|90%).These data clearly demonstrate the functional correction of the PAP (m|[szlig]|c-/-) phenotype by therapeutic m|[szlig]|c gene transfer. Furthermore, these studies advocate MGMTP140K as a suitable tool to enrich for therapeutic levels of genetically corrected cells in the context of hematopoietic stem cell gene therapy.