Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies.

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.

Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells.

Niemann-Pick type C disease (NP-C) is a progressive lysosomal lipid storage disease caused by mutations in the NPC1 and NPC2 genes. NPC1 is essential for transporting cholesterol and other lipids out of lysosomes, but little is known about the mechanisms that control its cellular abundance and localization. Here we show that a reduction of TMEM97, a cholesterol-responsive NPC1-binding protein, increases NPC1 levels in cells through a post-transcriptional mechanism. Reducing TMEM97 through RNA-interference reduces lysosomal lipid storage and restores cholesterol trafficking to the endoplasmic reticulum in cell models of NP-C. In TMEM97 knockdown cells, NPC1 levels can be reinstated with wild type TMEM97, but not TMEM97 missing an ER-retention signal suggesting that TMEM97 contributes to controlling the availability of NPC1 to the cell. Importantly, knockdown of TMEM97 also increases levels of residual NPC1 in NPC1-mutant patient fibroblasts and reduces cholesterol storage in an NPC1-dependent manner. Our findings propose TMEM97 inhibition as a novel strategy to increase residual NPC1 levels in cells and a potential therapeutic target for NP-C.

A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele.

Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.