Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice

Abstract

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. We have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy depends on the interaction of a fragment of insulin-like growth factor II (IGF-II), with the IGF-II binding site on the bifunctional, IGF-II cation-independent mannose 6-phosphate receptor. A chimeric protein containing a portion of mature human IGF-II fused to the C terminus of human beta-glucuronidase was taken up by MPS VII fibroblasts in a mannose 6-phosphate-independent manner, and its uptake was inhibited by the addition of IGF-II. Furthermore, the tagged enzyme was delivered effectively to clinically significant tissues in MPS VII mice and was effective in reversing the storage pathology. The tagged enzyme was able to reduce storage in glomerular podocytes and osteoblasts at a dose at which untagged enzyme was much less effective. This peptide-based, glycosylation-independent lysosomal targeting system may enhance enzyme-replacement therapy for certain human lysosomal storage diseases.

Fig. 1.

(A) Schematic depiction of the three enzyme preparations used in this study. The position of the four glycosylation sites in native hGUS are indicated by vertical lines. The two glycosylation sites that contain Man6-P (P) are indicated by filled circles. The positions of the signal peptides and GILT tag are indicated by open boxes. hGUS-GILT-F1 is hGUS-GILT that has been treated with the endoglycosidase F1, which removes most of the oligosaccharides, including all of the Man6-P. (B) SDS/PAGE of purified recombinant proteins used. (C) Uptake of hGUS, hGUS-GILT, and hGUS-GILT-F1 was studied as described in Methods. GM4668 cells were incubated with 4,000 units of each enzyme for 3 h in the presence or absence of 2 mM Man6-P (+M6P) or 2.86 mM IGF-II (+IGF-II). Media were removed, the cells were lysed, and GUS activity was determined. Each bar represents a determination from triplicate wells. The observed values of uptake for hGUS plus Man6-P and for hGUS-GILT-F1 plus IGF-II were <1.0 units/mg. (D) Determination of K_uptake. Enzyme concentrations ranging 1,000-80,000 units/ml were incubated for 2 h in MEM, supplemented with 2 mM l-glutamine and 15% FBS, and processed as described in Methods to generate uptake-saturation curves. A double-reciprocal Eadie-Hofstee plot determined the K_uptake for each recombinant enzyme. K_uptake was determined from titrations of the uptake of untagged hGUS (•) or hGUS-GILT-F1 (○) enzyme. Units on the x axis are uptake/input (U/I) or (mol/2)/mol. Units on the y axis are mol per 2 × 10¹⁰.

Fig. 2.

Biodistribution of hGUS, hGUS-GILT, and hGUS-GILT-F1 after a single injection into MPS VII mice. We infused 1 mg/kg of the indicated enzymes into six MPS VII mice for each treatment (n = 7 for hGUS-GILT) as described in Methods. After 24 h, the animals were killed, and tissue samples were processed for biochemistry, as described (11). Levels of enzyme observed in kidney, heart, and lung (A) or liver and spleen (B) are shown. Crosshatched bars, buffer control cells; black bars, hGUS; gray bars, hGUS-GILT; and white bars, hGUS-GILT-F1. Bars show the average of six to seven animals. Error bars indicate SD. (A) Student's t test, indicating that the differences between hGUS and hGUS-GILT observed in kidney, heart, and lung were statistically significant (P < 0.05).

Fig. 3.

Histochemical analysis of enzyme localization after a single injection. (A-C) After a single infusion of hGUS, enzyme activity was present primarily in the Kupffer cells (arrow), with only a small amount of activity in hepatocytes. Both hepatocytes (arrowheads) and Kupffer cells contain enzyme activity in the hGUS-GILT-treated and hGUS-GILT-F1 mice. (ASBI β-glucuronide, ×400 magnification.) (D) Glomeruli from a mouse treated with a single dose of hGUS had activity. (E and F) After hGUS-GILT and hGUS-GILT-F1 infusion, enzyme was present in the glomeruli in a similar distribution. (ASBI β-glucuronide, ×400 magnification.)

Fig. 4.

Reversal of storage after a short course of ERT. (A) The liver from an untreated MPS VII mouse had abundant storage in the Kupffer cells (arrow) and a small amount of storage in the hepatocytes. (B) After treatment with hGUS, there was a marked reduction in storage in both the hepatocytes and Kupffer cells (arrow). (C) A similar reduction in storage in hepatocytes and Kupffer cells (arrow) was present after hGUS-GILT treatment. (D) A glomerulus from an untreated MPS VII mouse had abundant lysosomal storage in the visceral epithelial cells (arrow). (E) After three injections of hGUS, lysosomal storage in glomerular visceral epithelial cells (arrow) was present in amounts similar to that seen in the untreated MPS VII mouse. (F) After treatment with hGUS-GILT, there was a reduction in storage in the glomerular visceral epithelial cells (arrow). (G) Osteoblasts (arrow) lining the bone of an untreated MPS VII mouse had lysosomal storage distending the cytoplasm. (H) After treatment with hGUS, the osteoblast (arrow) lysosomal storage persisted. (I) With hGUS-GILT treatment, the amount of lysosomal storage in osteoblasts (arrow) was markedly reduced. (A-C) Toluidine blue. (D-I) Uranyl acetate-lead citrate. [Magnifications, ×500 (A-C); ×1,428 (D-F); ×2,428 (G); and ×1,714 (H-I).]