In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins.
ABSTRACT Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human alpha-galactosidase (rhalphaGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in alphaGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution.
- Clinical Therapeutics 01/2009; 31. DOI:10.1016/S0149-2918(09)80013-1 · 2.59 Impact Factor
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ABSTRACT: Abstract A major driver of aging is catabolic insufficiency, the inability of our bodies to break down certain substances that accumulate slowly throughout the life span. Even though substance buildup is harmless while we are young, by old age the accumulations can reach a toxic threshold and cause disease. This includes some of the most prevalent diseases in old age-atherosclerosis and macular degeneration. Atherosclerosis is associated with the buildup of cholesterol and its oxidized derivatives (particularly 7-ketocholesterol) in the artery wall. Age-related macular degeneration is associated with carotenoid lipofuscin, primarily the pyridinium bisretinoid A2E. Medical bioremediation is the concept of reversing the substance accumulations by using enzymes from foreign species to break down the substances into forms that relieve the disease-related effect. We report on an enzyme discovery project to survey the availability of microorganisms and enzymes with these abilities. We found that such microorganisms and enzymes exist. We identified numerous bacteria having the ability to transform cholesterol and 7-ketocholesterol. Most of these species initiate the breakdown by same reaction mechanism as cholesterol oxidase, and we have used this enzyme directly to reduce the toxicity of 7-ketocholesterol, the major toxic oxysterol, to cultured human cells. We also discovered that soil fungi, plants, and some bacteria possess peroxidase and carotenoid cleavage oxygenase enzymes that effectively destroy with varied degrees of efficiency and selectivity the carotenoid lipofuscin found in macular degeneration.Rejuvenation Research 12/2009; 12(6):411-9. DOI:10.1089/rej.2009.0917 · 3.93 Impact Factor
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ABSTRACT: Fabry disease, due to the deficiency of α-galactosidase A (α-Gal), causes lysosomal accumulation of globotriaosylceramide (Gb3) in multiple tissues and prominently in the vascular endothelium. Although enzyme replacement therapy (ERT) by injection of recombinant α-Gal improves the disease outcome, the effects on the vasculopathy associated with life-threatening cerebrovascular, cardiac and renal complications are still limited. We designed a strategy to enhance the delivery of α-Gal to organs and endothelial cells (ECs). We targeted α-Gal to intercellular adhesion molecule 1 (ICAM-1), a protein expressed on ECs throughout the vasculature, by loading this enzyme on nanocarriers coated with anti-ICAM (anti-ICAM/α-Gal NCs). In vitro radioisotope tracing showed efficient loading of α-Gal on anti-ICAM NCs, stability of this formulation under storage and in model physiological fluids, and enzyme release in response to lysosome environmental conditions. In mice, the delivery of (125)I-α-Gal was markedly enhanced by anti-ICAM/(125)I-α-Gal NCs in brain, kidney, heart, liver, lung, and spleen, and transmission electron microscopy showed anti-ICAM/α-Gal NCs attached to and internalized into the vascular endothelium. Fluorescence microscopy proved targeting, endocytosis and lysosomal transport of anti-ICAM/α-Gal NCs in macro- and micro-vascular ECs and a marked enhancement of Gb3 degradation. Therefore, this ICAM-1-targeting strategy may help improve the efficacy of therapeutic enzymes for Fabry disease.Journal of Controlled Release 10/2010; 149(3):323-31. DOI:10.1016/j.jconrel.2010.10.031 · 7.26 Impact Factor