Intraperitoneal Injection of Genetically Modified,Human Mesothelial Cells for Systemic Gene Therapy

Abstract

An ideal cell type for ex vivo gene therapy should be easy to biopsy, propagate, and genetically engineer in culture, should be transplantable using simple procedures, and should express therapeutic proteins at useful levels. The mesothelial cell appears to satisfy these criteria. Several thousand proliferative mesothelial cells were present in typical specimens of nonpathologic human peritoneal fluid obtained by needle aspiration. These divided rapidly in a specialized medium to yield pure cultures of ∼10⁷ cells within 2 weeks. The replicative lifespan of mesothelial cells cultured from adults was ∼42–52 population doublings, permitting expansion and cryopreservation of a lifetime supply of autologous cells from one fluid sample. Cells transduced with a human growth hormone (hGH) adenoviral vector secreted 100–300 μg of hGH/10⁶ cells per day for at least 6 weeks in culture when maintained at quiescence. Intraperitoneal injection of transduced cells into athymic mice resulted in rapid systemic delivery of hGH, with peak plasma levels of 0.1–1 μg/ml declining over 3 weeks to <1 ng/ml. Mice receiving a second injection of engineered cells displayed the same plasma hGH levels and duration as naive mice. Cells labeled with a β-galactosidase vector were identifiable by in situ enzymatic staining as clusters attached to peritoneal surfaces at multiple sites for at least 19 days after injection. Cells serially passaged through about three-quarters of their lifespan before transduction and injection were as effective at hGH delivery as earlier-passage cells. These results indicate the clinical potential for ex vivo gene therapy using mesothelial cells.

Overview summary

The peritoneal cavity has long been recognized as being an excellent potential site for introducing genetically modified cells that secrete a therapeutic product targeted for the systemic circulation. The mesothelial cell, which forms the simple squamous epithelium that lines this cavity, should be an ideal cell type to use for this purpose. We have found that proliferative mesothelial cells are present in sufficient quantity in normal human peritoneal fluid, which is retrievable by needle aspiration, to permit the generation of large numbers of cells by expansion in culture. These cells were genetically engineered in culture by adenoviral transduction to secrete human growth hormone (hGH). When injected intraperitoneally as a cell suspension into athymic mice, they yielded high plasma levels of hGH for several weeks. These results demonstrate an effective system for ex vivo gene therapy using mesothelial cells.