SYSTEMIC DELIVERY OF HUMAN GROWTH-HORMONE OR HUMAN FACTOR-IX IN DOGS BY REINTRODUCED GENETICALLY-MODIFIED AUTOLOGOUS BONE-MARROW STROMAL CELLS

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfe cted to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adhere nt bone marrow stromal cell cultures, established and expanded from il iac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3. 84 mu g/10(6) cells per 24 hr hGH in vitro. The transfected plasmid ve ctor does not possess a eukaryotic origin of replication nor does it p ossess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases , the stromal cells were cryopreserved following transfection, and sub sequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, an d infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 m u g/24 hr. Plasma of each of the dogs contained detectable hGH for a m ean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following re infusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Ve ctor-modified stromal cells were detectable, by polymerase chain react ion (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were d etected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respective ly, following reinfusion. Tn another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid v ector. Modified cells (5.57 x 10(8)), with a total hFIX expression cap acity of 281 mu g/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.