In vitro retroviral vector-mediated transfer of the rat beta-glucuronidase cDNA into canine fetal liver cells and weanling MPS VII bone marrow cells

A beta-glucuronidase cDNA was transferred into normal canine fetal liver cells and bone marrow cells obtained from weanling dogs affected with mucopolysaccharidosis (MPS) type VII. The cells were transduced by direct cocultivation with a recombinant retrovirus-producing. cell line, and by culturing the hematopoietic cells and vector packaging cells separated by a 0.45 mu m filter in a dual-chambered cocultivation system. The weanling MPS VII dog bone marrow cells were also transduced in long-term cultures with high titer vector virus. Gene transduction was achieved in all fetal liver cells obtained from 24 and 35 day old dog fetuses, but not 55 day old fetuses. The post-natal bone marrow cells were transduced by each of these methods, The results indicate that early gestation fetal liver cells can be transduced in culture conditions without contaminating virus-producing packaging cells. Mucopolysaccharidosis type VII (MPS VII) in humans, mice, and dogs is a well characterized progressive, genetic disease caused by a deficiency of beta-glucuronidase (GUSB) activity.(1-4) Microscopic signs of disease are present at birth,(5) thus the animal models can be used to determine if providing GUSB activity at an early age or in utero reduces the pathology associated with MPS VII. Bone marrow transplantation of hematopoietic stem cells (HSC) provides a means of treating a variety of lysosomal storage diseases by providing a source of normal enzyme.(4,6) 6 However, the efficacy of bone marrow transplantation is variable depending on the type of storage disease and the age at which transplantation is performed.(6-8) In addition, matched donors frequently are not available and the recipient must be conditioned either by irradiation or chemotherapy, both of which bear the risk of diseases associated with these pretransplantation treatments. Potential complications of bone marrow transplantation are graft rejection and graft versus host disease.(9) Many of the side effects of bone marrow transplantation may be avoided by transferring a functional exogenous gene into autologous hematopoietic stem cells in bone marrow. Retroviral vectors efficiently transfer exogenous genes into murine hematopoietic stem cells.(10-14) However, transplantation of transduced hematopoietic stem cells into adults may not lead to reversal of all aspects of disease.(14) In addition, studies in larger animal species have shown that efficiency of transduction and expression of the transferred gene are low.(15-17) Introduction of an exogenous gene into fetal or neonatal hematopoietic stem cells may result in higher transduction rates and better expression. In utero intervention may reduce the pathology caused by the gene defect, or prevent manifestation of disease altogether In this study we used a retroviral vector containing GUSB cDNA that has been shown to transduce adult hematopoietic stem cells(14) and express GUSB in affected canine cells.(18-20) This study examined retroviral transduction of canine fetal liver cells of 24, 35, and 55 days gestational age, and canine MPS VII bone marrow cells obtained 42 days post-natally.