Krabbe Disease: Prospects of Finding a Cure Using AAV Gene Therapy

Krabbe Disease (KD) is an autosomal metabolic disorder that affects both the central and peripheral nervous systems. It is caused by a functional deficiency of the lysosomal enzyme, galactocerebrosidase (GALC), resulting in an accumulation of the toxic metabolite, psychosine. Psychosine accumulation affects many different cellular pathways, leading to severe demyelination. Although there is currently no effective therapy for Krabbe disease, recent gene therapy-based approaches in animal models have indicated a promising outlook for clinical treatment. This review highlights recent findings in the pathogenesis of Krabbe disease, and evaluates AAV-based gene therapy as a promising strategy for treating this devastating pediatric disease.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese Hamster Ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-a-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with 9 different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igk light chain and human chymotrypsinogen B1 signal peptides, whereas Igk light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Fabry disease: a case report

Fabry disease is a rare X-linked recessive inborn error of metabolism due to deficient activity of the lysosomal enzyme, a-galactosidase A (a-Gal A). This results in the tissue accumulation of uncleaved glycosphingolipids within vascular endothelial lysosomes of various organs including skin, heart, kidneys and brain. We report a case of Fabry disease, in an 18-year-old boy, who presented with unilateral leg swelling and angiokeratoma corporis diffusum. Birdem Med J 2021; 11(2): 145-147

Optimization of the Cultivation Processof a Producer Clone of the Recombinant Lysosomal Iduronate-2-sulfatase Enzyme

This paper describes experiments aimed at developing conditions for suspension culturing of a producer clone of the recombinant lysosomal enzyme iduronate-2-sulfatase based on the CHO cell line. As a result of sequential culturing in batch and fed-batch modes, the optimal nutrient medium composition was established, providing a 2.5-fold increase in the iduronate-2-sulfatase specific activity. Optimization of the feed formulation was carried out, which increased the yield of recombinant iduronate-2-sulfatase. The ambr® 15 Cell Culture System of minibioreactors was used to optimize the cultivation process. Chinese hamster ovary cells, CHO, iduronate-2-sulfatase, idursulfase, lysosomal enzyme, sulfatases, Аmbr Tap Biosystems, ambr® 15 Cell Culture System, mucopolysaccharidosis type II, formyl glycine generating enzyme, optimization of cell culturing conditions, fed-batch culture, batch culture.

Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game

Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of...

Alleviated Immunological Activities of Wistar Rat Peritoneal Neutrophils and Macrophages by Polysaccharide Rich Extract from the Fruit Bodies of Microporus vernicipes (Polyporales)

Aim: This study aimed to assess the effect of polysaccharides from fruit body of Microporous vernicipes (Polyporales) on immunological activities of macrophages and neutrophils. Methodology: Polysaccharides were extracted using hot water (PHW) and cool water (PCW) and tested on the activities of peritoneal macrophage and neutrophil in response of lipopolysaccharides. The effect of the polysaccharides was examined In vitro to assess the phagocytic activities by evaluating the vacuole formation, nitroblue tetrazolium dye reduction, lysosomal enzyme activity, nitric oxide, myeloperoxidase in neutrophil and on neutrophil adherence assay. Results: The cool and hot water polysaccharides of Microporus vernicipes significantly improved the activities of macrophages and neutrophils as demonstrated by an elevated the formation of vacuole following by a high production of nitric oxide, lysosomal enzyme and superoxide. Those polysaccharides extracts of Microporous vernicipes also caused an increase in release of myeloperoxidase by neutrophils. Results also showed that neutrophils treated with polysaccharides extracts of Microporous vernicipes adhered more on a plastic surface than those untreated. It has been also found that polysaccharides of Microporous vernicipes may have cytotoxic effect at high concentration with IC50 of 2.28 and 8.15 mg/mL for PHW and PCW respectively. Conclusion: The result of this study indicates the immunostimulatory activity of polysaccharides of Microporus vernicipes by stimulating activities of macrophages and neutrophils.