Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α-<i>N</i>-Acetylglucosamine-6-Sulfatase in Neonatal Mice

Wang, Rui; Moen, Derek R.; Sauni, Chelsee; Kan, Shih Hsin; Li, Shan; Le, Steven Q.; Lomenick, Brett; Zhang, Xiaoyi; Ekins, Sean; Singamsetty, Srikanth; Wood, Jenny; Dickson, Patricia I.; Chou, Tsui Fen

doi:10.1021/acs.molpharmaceut.0c00831

Cited by 11 publications

(9 citation statements)

References 98 publications

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“…In addition, the increases in LDLR, APOB and NAGLU were also observed from our previous proteomic data (Figure S1B) [16]. To validate our proteomic data, we used enzymatic activity assays to measure the active enzyme levels of NAGLU and HEXB [23,24]. Our results showed UPCDC-30245 treated samples have significant increase of HEXB and NAGLU enzymatic activities (Figure 2E), and we demonstrated that UPCDC-30245 did not affect their activities when added directly to cell lysate (Figure 2E), which is consistent with the increased protein levels in our proteomic data.…”

Section: Proteomics Reveals Impairment Of Endo-lysosomal Pathways In ...supporting

confidence: 76%

“…Therefore, the reduction of early endosomes is a cellular effect potentially unique to UPCDC-30245. The upregulation of LAMP1, lysosomal enzymes (HEXB and NAGLU) as well as autophagy has been found in multiple lysosome storage diseases (LSDs) [23,24,49]. LSDs are caused by the insufficient degradation of (GAGs) due the deficiency of certain lysosome enzymes.…”

Section: Discussionmentioning

confidence: 99%

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The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

Wang

Cheng

et al. 2022

Pharmaceuticals

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The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry.

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Section: Proteomics Reveals Impairment Of Endo-lysosomal Pathways In ...supporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

Wang

Cheng

et al. 2022

Pharmaceuticals

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“…Both rAAV‐mediated gene and enzyme replacement therapies have been applied to TPP1 deficiency (Katz et al, 2015). Intracranial delivery of rAAV has shown an exponential increase in TPP1 enzyme activity, while enzyme replacement therapy (ERT) has shown an improvement in the symptomatology and neuropathology of the disease, both in vitro using human fibroblast and in vivo using murine models and non‐human primates (Wang et al, 2021).…”

Section: Murine Modelsmentioning

confidence: 99%

Batten disease through different in vivo and in vitro models: A review

Nittari

Tomassoni

Roy

et al. 2022

J of Neuroscience Research

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Batten disease consists of a family of primarily autosomal recessive, progressive neuropediatric disorders, also known as neuronal ceroid lipofuscinoses (NCLs). These pathologies are characterized by seizures and visual, cognitive and motor decline, and premature death. The pathophysiology of this rare disease is still unclear despite the years of trials and financial aids. This paper has reviewed advantages and limits of in vivo and in vitro models of Batten disease from murine and larger animal models to primitive unicellular models, until the most recently developed patient‐derived induced pluripotent stem cells. For each model advantages, limits and applications were analyzed. The first prototypes investigated were murine models that due to their limits were replaced by larger animals. In vitro models gradually replaced animal models for practical, cost, and ethical reasons. Using induced pluripotent stem cells to study neurodegeneration is a new way of studying the disease, since they can be distinguished into differentiating elements like neurons, which are susceptible to neurodegeneration. In vivo and in vitro models have contributed to clarifying to some extent the pathophysiology of the disease. The collection and sharing of suitable human bio samples likely through biobanks can contribute to a better understanding, prevention, and to identify possible treatment strategies of Batten disease.

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“…The human full-length NAGLU cDNA (amino acid 1-743) in pEE12.4/NAGLU-IGFII with a C-terminal myc tag (25) was subcloned into pOptiVEC™-TOPO® shuttle vector (Invitrogen), bearing a gene coding for dihydrofolate reductase (DHFR). A human full-length GNS cDNA (amino acid 1-552) followed by a myc tag was subcloned into pOptiVEC™-TOPO® vector as described (26). To construct rhGNS with an artificial signal peptide, the signal peptides followed by a XhoI site were fused at the 5' primer of the GNS coding sequence.…”

Section: Plasmid Dna Constructionmentioning

confidence: 99%

“…NAGLU or GNS activity was measured in triplicate by detecting the amount of 4-methylumbelliferon (4-MU) to be liberated from the respective fluorogenic substrate for both GNS and NAGLU (25,26).…”

Section: Enzymatic Activity Assays For Rhnaglu and Rhgnsmentioning

confidence: 99%

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

Cheng

Wang

Lopez

et al. 2021

Biochemical Journal

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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.

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Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α-N-Acetylglucosamine-6-Sulfatase in Neonatal Mice

Cited by 11 publications

References 98 publications

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

Batten disease through different in vivo and in vitro models: A review

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

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