Background: Acid α-glucosidase, an enzyme replacement therapy for Pompe disease, is poorly targeted to lysosomes when relying on phosphomannose residues.Results: Fusing IGF-II to acid α-glucosidase resulted in more efficient uptake and glycogen clearance from muscle of Pompe mice.Conclusion: Enhanced binding to the cation-independent mannose 6-phosphate receptor (CI-MPR) enabled improved glycogen clearance in Pompe mice.Significance: BMN 701 is now being tested for Pompe disease in human clinical studies.

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 cellsurface 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 ␤-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.␤-glucuronidase ͉ IGF-II͞Man6-P receptor ͉ receptor-mediated endocytosis ͉ enzyme-replacement therapy ͉ lysosomal storage disease

Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.

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