870. Gene Transfer in the Sandhoff Murine Model Using Lentiviral Vectors

Abstract

The Sleeping Beauty (SB) transposon system created at the University of Minnesota is one of the few non-viral gene therapy systems that are able to integrate genes into human chromosomes. This is especially relevant for treating genetic disorders that require sustained expression of the therapeutic transgene. The purpose of this study is to evaluate SB-mediated transposition as a tool for safe, efficient, stable, non-viral gene therapy in a model lysosomal disorder, mucopolysaccharidosis (MPS) type VII. Cis SB transposon plasmids were constructed to deliver both the T2 transposon and SB11 transposase on the same plasmid (Geurts et al., Mol. Ther. v. 8, p. 108-17, 2003). For MPS VII, we constructed a transposon carrying the human -glucuronidase (GUSB) cDNA driven by a strong ubiquitous CAGGS promoter, and with SB11 transcribed from the intermediate-strength ubiquitin C promoter. Expression levels of SB11 in murine liver were confirmed by Western analysis. MPS VII mice completely deficient in GUSB activity were hydrodynamically injected with 25 g of plasmids that either did, or did not, express SB11. Blood samples for plasma isolation were collected 24 h after treatment and biweekly thereafter. An injection was deemed technically successful by the observation of plasma GUSB levels >100-fold of wild type levels 24 h following treatment. Four weeks after treatment, all plasma GUSB activities were below the lowest level of detection (probably not due to immune response because Bethesda Assays did not indicate the presence of GUSB inhibitors). Mice were euthanized 3 months after treatment. At that time GUSB activity in liver homogenates from mice that received transposase was 10-fold higher compared to that in the no-transposase control group, which was equal to approximately 1% of the value for normal untreated mice. Transgene frequency (as estimated by GUSB histochemical staining of frozen liver sections) was below 0.1% in the former group, but virtually undetectable in mice receiving transposon without transposase. Toluidine blue staining of liver sections was used to evaluate whether the amount of enzyme expressed from the transposons was sufficient to reduce the number and size of pathologic lysosomal inclusion in livers. GUSB activity levels in the spleen, lung, and kidney were 1% wild type levels and also produced transient expression of exceedingly high levels of circulating GUSB enzyme. These studies define a pattern of biodistribution and transgene expression that may be therapeutic for other MPS diseases in addition to MPS VII.