Adenovirus-mediated gene transfer and expression of human beta-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice

Abstract

Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme beta-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human beta-glucuronidase and administered this recombinant adenovirus to beta-glucuronidase-deficient mice intravenously. The beta-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the beta-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the beta-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of beta-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology.

Figure 1

Recombinant adenovirus AxCAHBG. The HBG cDNA was cloned downstream of the CAG (cytomegalovirus enhancer-chicken β-actin hybrid) promoter in pAxCAwt. A rabbit β-globin poly(A) sequence was located downstream of the HBG cDNA.

Figure 2

Histochemical staining for β-glucuronidase activity in the liver of an untreated MPS VII mouse (A and B) shows no enzyme activity. Sixteen days after receiving 1.79 × 10⁹ pfu of recombinant adenovirus, the liver from an MPS VII mouse (C and D) shows numerous β-glucuronidase-containing cells. Their distribution and location suggest that many are sinus lining cells. Scattered large polygonal cells morphologically consistent with hepatocytes also contained β-glucuronidase. Thirty-five days after receiving 1.79 × 10⁹ pfu of recombinant adenovirus (E and F), β-glucuronidase-containing cells are still seen, although they are less numerous than at 16 days after injection. (A, C, and E, ×20; B, D, and F, ×210.)

Figure 3

The ependyma (solid arrow) and choroid lining (open arrow) of the third ventricle (A and B) contained histochemically demonstrable β-glucuronidase activity 15 days after 4.48 × 10⁸ pfu of recombinant adenovirus was injected into the lateral ventricle. In the same mouse, scattered β-glucuronidase-positive cells were also seen in cells associated with vessels (C) in the parenchyma. The meninges (D) contained perivascular and parenchymal cell β-glucuronidase activity. (A, ×50; B, ×210; C and D, ×130.)

Figure 4

The liver of an untreated, age-matched MPS VII mouse (A) showed lysosomal distention in Kupffer cells and a lesser amount of storage in hepatocytes. Thirty-five days after receiving recombinant adenovirus (B), both hepatocyte storage and Kupffer cell storage were markedly reduced. The untreated MPS VII mouse spleen (C) contained abundant lysosomal storage in sinus lining cells in the red pulp. Thirty-five days after recombinant adenovirus (D), there was a similar marked reduction in the amount of lysosomal storage. (A–D, toluidine blue; A–D, ×190.)