INTRAMARROW CYTOKINE GENE-TRANSFER BY ADENOVIRAL VECTORS IN DOGS

Daily systemic administration of hematopoietic growth factors can be a ssociated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marro w cavity of dogs by direct intramarrow injection of adenoviral vectors . In marrow culture, replication-deficient (E1-deleted) adenoviral vec tors were able to transduce marrow stromal cells, demonstrating 30-fol d greater expression than from other marrow cell types. High-level (ng /ml) cytokine production from transduced stromal cells persisted for 1 4 days in culture. Because adenovectors could efficiently transduce ma rrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injecti on. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-g alactosidase) were injected directly into the marrow cavity of dogs re sulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector exp ressing biologically active canine granulocyte-macrophage colony stimu lating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bi oactivity of protein expressed was confirmed by canine GM colony-formi ng assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow ad ministration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-h r sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow a nd significant peripheral leukocytosis (neutrophilia) were noted in al l ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 w eeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of h ematopoiesis sufficient to cause peripheral blood changes in a large a nimal model.