854. Feasibility of AAV-Mediated Gene Therapy Examined Using a New Murine Model (D409V/null) of Gaucher Disease

Abstract

Gaucher disease is the most common of the more than 40 currently described lysosomal storage diseases. It is caused by mutations in the gene encoding the lysosomal hydrolase, glucocerebrosidase (GC). The loss or deficiency of this enzyme results in the accumulation of the substrate, glucosylceramide (GL-1), in tissue macrophages primarily of the liver and spleen. A mouse model of the disease (D409V/null) was recently generated. Characterization of this murine model indicated that it displayed several of the biochemical and phenotypic abnormalities shown associated with the human disease. The D409V/null mice exhibited approximately only 5% of normal levels of the enzyme in the visceral tissues and consequently, elevated levels of GL-1 in the liver, spleen, lung and also the bone marrow of these animals. Associated with the abnormal storage of GL-1 in the tissues were the appearance of characteristically enlarged macrophages (Gaucher cells) similar to those observed in Gaucher patients, particularly in the liver, spleen and lung of the mice. To evaluate the potential of gene therapy for treating this disease, an AAV 2/2 and 2/8-pseudotyped vector encoding the human GC were constructed. Since secretion of GC from transduced cells occurs only in cells highly overexpressing the enzyme, efforts were made to optimize its expression. A codon optimized and CpG-reduced synthetic cDNA for human GC was placed under the transcriptional control of an 1-antitrypsin promoter to which was appended two copies of the 1-microglobulin enhancer (DC172). Studies showed that expression from the DC172 promoter was hepatic-restricted and was significantly higher than that attained with either a CMV or the previously described DC190 promoter (human serum albumin promoter linked to two copies of the human prothrombin enhancer). Intravenous administration of 31011 particles of either AAV2/2-DC172-shGC or AAV2/8-DC172-shGC into 4 weeks old D409V/null mice resulted in hepatic transduction and subsequent secretion of supraphysiological levels of GC into the systemic circulation. Expression of GC in animals administered AAV2/8-DC172-hGC were 50 to 100-fold higher than from the corresponding AAV2/2 vector and remained undiminished at 4 months. In contrast to the untreated mice, which displayed characteristic Gaucher cells in the lungs, liver and spleen, treated animals (at 2 months post-treatment) were devoid of these cells and harbored normal tissue levels of GL-1. The absence of elevated GL-1 levels in the tissues of treated animals confirmed that the hepatically-produced enzyme was in a form that was conducive to recapture by the affected cells and that importantly, the levels attained were sufficient to prevent the development of disease manifestations. These data support the continued development of AAV vectors for gene therapy of Gaucher disease.