Intramarrow Cytokine Gene Transfer by Adenoviral Vectors in Dogs

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Abstract
Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (β-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating 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 bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean ± SEM, 27 ± 22 ng/ml, 72-hr sample 921 ± 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks 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 hematopoiesis sufficient to cause peripheral blood changes in a large animal model. Hematopoiesis takes place in bone marrow and involves the continuous production of erythrocytes, leukocytes, and platelets. Hematopoiesis can be significantly influenced by growth factor cytokines. Some growth factors are locally produced from stromal cells within the bone marrow. Growth factors are also used in clinical practice and are given by systemic injection. Recombinant growth factors can enhance hematopoiesis, and they are useful in a variety of clinical settings. Daily subcutaneous administration of growth factors can be associated with dose-limiting systemic side effects, and, as an alternative approach, we demonstrate that local tissue-specific high-level expression of cytokine growth factors can be largely confined to within the marrow space. This is achieved by adenoviral gene transfer to marrow stromal cells. In this approach, vector is injected into the marrow cavity, resulting in transduction of stromal cells and local expression of cytokine protein. In this study, adenoviral cytokine gene transfer and stromal overexpression of growth factors is evaluated in long-term bone marrow culture and following direct intramarrow vector administration in dogs.