Gene therapy of lysosomal storage diseases, including MPS, requires sustained expression of the therapeutic gene. Non-viral vector systems have been questioned ‘as a stand-alone therapy’ for these disorders, in part due to the transient nature of plasmid-based expression of the therapeutic gene [42
]. The Sleeping Beauty
(SB) transposon system combines the ability to integrate genes into chromosomes for prolonged expression with the advantages of plasmid-based vectors. Ease of formulation, quality control, storage and shipment are characteristics that will be important for treating significant populations of patients.
Expression of hIDUA
from a plasmid-based vector in MPS I mouse for 14 days has been recently reported [43
]. This is not customarily considered ‘long-term’, because transient activity persists over this period. However, our goal was prolonged IDUA expression. In the same mouse model, our study demonstrates for the first time a high level of expression lasting up to more than 3 months (). In adult immunomodulated MPS I mice, delivery of the SB transposon and transposase-encoding plasmid to the liver by rapid tail-vein injection resulted in persistent expression of human IDUA enzyme in one-third of the treated animals. Notably, in all but one mouse, the sustained IDUA activities in plasma were high, ranging between WT and 100-fold WT levels. In contrast, in the absence of transposase, only about 20% of the immunosuppressed mice exhibited maintained expression, and these levels did not exceed 40% WT. Our assay for the excision product (EP, ) demonstrated that transposition activity mobilized IDUA transposons in mouse liver. The transgene copy number in cyclophosphamide-immunosuppressed mice (e.g., mouse 2, Figures and ) was up to 7 per 100 cells, resulting in the treated liver expressing hIDUA at WT activity level. In mouse 1 that exhibited reduced IDUA activity at 14 weeks post-injection (Figures and ), only about 1% of cells contained transgenes at the time of analysis. Neither transgenes nor transgene products were detected without immunomodulation (mouse 3, Figures and ).
Our studies with MPS VII mice allowed us to determine the efficiency of gene transfer to hepatocytes by hydrodynamic injection, which was up to 15–30% (), similar to the results of Yant et al
] and Wilber et al
]. Eight weeks post-injection, about 5–10% of transfected liver cells in one treated mouse () were still able to provide enzymatic cross-correction to the whole liver, yielding GUSB activity in total liver homogenates equal to about 13% WT activity and complete or close to complete correction of lysosomal pathology. In other mice, approximately 2% and 0.5% transfected cells yielded GUSB activity in the liver about 3% and 1% WT, respectively, which resulted in partial correction of liver pathology. In adult MPS I mice, prolonged IDUA expression at WT levels reduced storage in parenchymal organs with complete and near-complete correction of storage in liver and spleen, respectively.
Recent advances in preclinical gene therapy of MPS I have been impressive. In adult MPS I mice, Zheng et al
] and Jordan et al
] demonstrated significant biochemical and histological improvements, as well as normalization of ventricular function after transplantation with retrovirally transduced syngeneic bone marrow. Using either adeno-associated virus [21
] or lentiviral vector [23
] to deliver therapeutic IDUA genes to newborn mice, persistent high-level IDUA expression over several months was observed in the liver, heart, lung and brain with subsequent curative impact on several of the most important parameters of the disease, including reduced storage vacuoles in a number of tissues, reduced glycosaminoglycan excretion, prevention of craniofacial abnormalities and behavioral abnormalities. Using a single injection of retroviral vector in neonates Liu et al
] demonstrated sustained supra-normal IDUA expression in the liver throughout an 8-month experiment which resulted in prevention of major clinical manifestations of MPS I including cardiac and bone disease, hearing loss and vision abnormalities. The latter study demonstrated the effectiveness of targeting hepatocytes. Stable serum IDUA activities at about 10-fold WT were partially curative, but with 100-fold WT levels, profound correction of MPS I disease was achieved. Our most important finding is that in adult mice we can achieve sustained expression in the liver at levels comparable to those shown in studies that used viral vectors. Our results using a transposon-mediated approach show that with immunomodulation the sustained transgenic IDUA activity in adult mice can reach as high as 100-fold WT level. However, we were unable to prevent immune responses completely in all mice. This is the most likely explanation for the wide variation of transgenic IDUA activities that we observed in mice treated with the complete transposon system (). With a better protocol for immunomodulation, we expect to attain predictably high IDUA expression levels in all mice.
The surprising finding that animals treated with just a transposon carrying hGUSB outperformed those that were treated with the complete SB system () was the first suggestion that some biological response was suppressing prolonged expression of newly synthesized hGUSB. Although the number of animals that maintained GUSB activity 8 weeks post-injection (n = 2) was too small to be significant, we speculate that liver cells exhibiting prolonged high hGUSB expression levels due to transgene integration elicited a strong immune response and were eliminated, while the lower expression levels from unintegrated plasmid were insufficient to induce an equally efficient immune response.
Cell-mediated immune responses can be expected, particularly when the hydrodynamic approach results in overexpression of the transgenic protein regulated by a strong promoter. Although this technique delivers DNA primarily to the liver and expression levels in other organs are low (1–0.1% those in the liver [31
], and Dr. Andy Wilber, University of Minnesota, personal communication), equally high levels of transgenic IDUA or GUSB enzyme, exceeding WT levels in these organs by over 10-fold, persist in the spleen, for almost a week post-injection (not shown). This could expose antigen-presenting cells both in the liver and in the spleen to massive amounts of transgenic enzyme.
Transgene-specific host immune responses following somatic gene transfer, both humoral and cell-mediated, have been described (e.g., [47
]). Our study affirms that this serious issue needs to be addressed even in the case of naked DNA delivery. Without immunomodulation, liver cells that harbored the transgene disappeared for the most part by 4–6 weeks post-injection, with or without SB transposase. This loss was supported by observation of a low number of small inflammatory cell infiltrates composed of predominantly mononuclear cells and a few neutrophils surrounding either morphologically apoptotic/necrotic hepatocytes or round holes, ‘hepatocyte dropouts’ in the livers of plasmid-injected mice, but not in PBS-injected mice. The onset of infiltrates appeared to be time-dependent. Only some of the mononuclear cells in the infiltrates were CD4- and CD8-positive lymphocytes. Other types of mononuclear cells within these inflammatory foci have yet to be characterized. The presence of neutrophils may be due to their attraction to the sites of cellular necrosis.
Elucidating the mechanisms by which transgenic cells are eliminated will be needed to develop approaches that will allow their retention. With validation that the SB transposon system can provide long-term correction of MPS diseases without the use of viral vectors, we now must develop methods for counteracting immune and other protective responses of the host. In this context the prospects for non-viral gene therapy of MPS diseases are quite encouraging.