Systemic Delivery of Human Growth Hormone or Human Factor IX in Dogs by Reintroduced Genetically Modified Autologous Bone Marrow Stromal Cells

Abstract

Canine bone marrow stromal cells were expanded to numbers in excess of 10⁹ cells from the initial 10–20 ml of marrow aspirates and transfected 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. Adherent bone marrow stromal cell cultures, established and expanded from iliac 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 μg/10⁶ cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess 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 subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 × 10⁷ to 2.6 × 10⁹, with total hGH expression capacities ranging from 62 to 1,400 μg/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD ± 0.8 day) ranging from 2 to 5 days following reinfusion 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. Vector-modified stromal cells were detectable, by polymerase chain reaction (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 detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 × 10⁸), with a total hFIX expression capacity of 281 μg/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. Ex vivo, autologous, somatic cell gene therapy involves the removal of cells from individuals, the genetic engineering of these cells to express transgene products, and their reintroduction into each individual. We have developed an ex vivo bone marrow stromal cell system to deliver secreted products to the systemic circulation of dogs. This report demonstrates that reinfused modified stromal cells deliver detectable levels of human growth hormone (for 2–5 days) or human factor IX (for 9 days) to the plasma of host dogs. PCR analyses demonstrated that modified stromal cells were detected in the circulation of all 4 dogs analyzed following infusion. In 3 of the 4 dogs, cells were detected for from 8.5 to 15 weeks. The bone marrow of infused dogs was similarly analyzed starting 4 weeks following infusion. In 2 of the 3 dogs analyzed, modified stromal cells were detected in bone marrow for 9 and 13 weeks, respectively. These results demonstrate that the bone marrow stromal cell system has potential value in gene therapy applications.