Identifying therapeutic drug targets using bidirectional effect genes.

Prioritizing genes for translation to therapeutics for common diseases has been challenging. Here, we propose an approach to identify drug targets with high probability of success by focusing on genes with both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). We find 98 BEST genes for a variety of indications. Drugs targeting those genes are 3.8-fold more likely to be approved than non-BEST genes. We focus on five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants in those genes result in significantly increased risk for idiopathic short stature (ISS) (OR = 2.75, p = 3.99 × 10-8). Finally, using functional experiments, we demonstrate that adding an exogenous CNP analog (encoded by NPPC) rescues the phenotype, thus validating its potential as a therapeutic treatment for ISS. Our results show the value of looking for bidirectional effects to identify and validate drug targets.

Intermittent enzyme replacement therapy with recombinant human ß-galactosidase prevents neuraminidase 1 deficiency

Mutations in the galactosidase ß 1 (GLB1) gene cause lysosomal ß-galactosidase (ß-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. ß-Gal and neuraminidase 1 (NEU1) form a multienzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, ß-Gal only partially depends on PPCA, prompting us to investigate the role that ß-Gal plays in the multienzyme complex. Here, we demonstrate that ß-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human ß-Gal (rhß-Gal) or chronic lentiviral-mediated GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy dosing with rhß-Gal, followed by enzyme withdrawal, is sufficient to augment ß-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of ß-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular dosing with rhß-Gal. Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of ß-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent intracerebroventricular enzyme replacement therapy dosing with rhß-Gal is a tunable approach that can safely augment ß-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain.

Intracerebroventricular enzyme replacement therapy with ß-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Autosomal recessive mutations in the galactosidase ß1 (GLB1) gene cause lysosomal ß-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human ß-gal (rhß-gal) produced in Chinese hamster ovary cells enabled direct and precise rhß-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhß-gal was sufficient for normalizing ß-gal activity and mediating substrate clearance for several weeks. We found that rhß-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor-mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhß-gal (100 µg) resulted in broad bilateral biodistribution of rhß-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhß-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of ß-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhß-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.

BMN 250, a fusion of lysosomal alpha-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis.

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.