Comparative transport analysis of cell penetrating peptides and Lysosomal sequences for selective tropism towards RPE cells.

Cell penetrating peptides are typically nonspecific, targeting multiple cell types without discrimination. However, subsets of Cell penetrating peptides (CPP) have been found, which show a 'homing' capacity or increased likelihood of internalizing into specific cell types and subcellular locations. Therapeutics intended to be delivered to tissues with a high degree of cellular diversity, such as the intraocular space, would benefit from delivery using CPP that can discriminate across multiple cell types. Lysosomal storage diseases in the retinal pigment epithelium (RPE) can impair cargo clearance, leading to RPE atrophy and blindness. Characterizing CPP for their capacity to effectively deliver cargo to the lysosomes of different cell types may expand treatment options for lysosomal storage disorders. We developed a combinatorial library of CPP and lysosomal sorting signals, applied to ARPE19 and B3 corneal lens cells, for the purpose of determining cell line specificity and internal targeting. Several candidate classes of CPP were found to have as much as 4 times the internalization efficiency in ARPE19 compared to B3. Follow-up cargo transport studies were also performed, which demonstrate effective internalization and lysosomal targeting in ARPE19 cells.

Fenretinide binding to the lysosomal protein saposin D alters ceramide solubilization and hydrolysis.

Fenretinide is a synthetic retinoid pharmaceutical linked to ceramide build-up in vivo. Saposin D is an intralysosomal protein necessary for ceramide binding/degradation. We show, via electronic absorption spectroscopy, fluorescence spectroscopy, and ceramide hydrolysis assays, that fenretinide is bound by saposin D {K a = (1.45 ± 0.49) × 105 M-1}, and affects ceramide solubilization/degradation.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

Saposin B Binds the Lipofuscin Bisretinoid A2E and Prevents its Enzymatic and Photooxidation.

Vitamin A based bisretinoid accumulation is a major focus in the study of macular degeneration. Whether specific endogenous lysosomal proteins can bind A2E, a pronounced bisretinoid in lipofuscin granules in retinal pigment epithelial cells, and interfere with enzymatic or photoinduced oxidation of such, has not been explored. Herein, using fluorescence and electronic absorption spectroscopy and mass spectrometry, we demonstrate that Saposin B, a critical protein in the degradation of sulfatides and "flushing" of lipids, can bind A2E, preventing its H2O2-dependent enzymatic oxidation by horseradish peroxidase and photooxidation by blue light (λ=450-460 nm).