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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. Prioritising genes as potential drug targets is challenging and often unsuccessful once testing efficacy in humans. Here, the authors propose an approach to identifying drug targets that uses evidence from gain- or loss-of-function mutations associated with bidirectional effects on phenotypes. [ABSTRACT FROM AUTHOR]

Difficult‐to‐express (DTE) recombinant proteins such as multi‐specific proteins, DTE monoclonal antibodies, and lysosomal enzymes have seen difficulties in manufacturability using Chinese hamster ovary (CHO) cells or other mammalian cells as production platforms. CHO cells are preferably used for recombinant protein production for their ability to secrete human‐like recombinant proteins with posttranslational modification, resistance to viral infection, and familiarity with drug regulators. However, despite huge progress made in engineering CHO cells for high volumetric productivity, DTE proteins like recombinant lysosomal sulfatase represent one of the poorly understood proteins. Furthermore, there is growing interest in the use of microRNA (miRNA) to engineer CHO cells expressing DTE proteins to improve cell performance of relevant bioprocess phenotypes. To our knowledge, no research has been done to improve CHO cell production of DTE recombinant lysosomal sulfatase using miRNA. We identified miR‐23a and miR‐377 as miRNAs predicted to target SUMF1, an activator of sulfatases, using in silico prediction tools. Transient inhibition of CHO endogenous miR‐23a/miR‐377 significantly enhanced recombinant sulfatase enzyme‐specific activity by ~15–21% compared to scramble without affecting cell growth. Though inhibition of miR‐23a/miR‐377 had no significant effect on the mRNA and protein levels of SUMF1, overexpression of miR‐23a/377 caused ~30% and ~27–29% significant reduction in endogenous SUMF1 protein and mRNA expression levels, respectively. In summary, our data demonstrate the importance of using miRNA to optimize the CHO cell line secreting DTE recombinant lysosomal sulfatase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB). Sanfilippo Type B is caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60,000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown. To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence. [ABSTRACT FROM AUTHOR]

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. The authors have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII model. This strategy depends on the interaction of a fragment of insulin-like growth factor II (IGF-II), with the IGF-II binding site on the bifunctional, lGF-II cation-independent mannose 6-phosphate receptor.

The article discusses the study "Overcoming the Blood-Brain Barrier With High-Dose Enzyme Replacement Therapy in Murine Mucopolysaccharidosis VII" by Carole Vogler and colleagues, published in the October 2005 issue of the journal "Proceedings of the National Academy of Sciences of the United States of America." A fundamental problem affecting the efficacy of drugs is their ability to be delivered from the site of administration to the site of pathology. This study provides an encouraging demonstration that a therapeutic lysosomal enzyme can be delivered across the blood-brain barrier (BBB), thereby reversing storage in brain tissue. Overcoming the BBB can be seen as the latest in a series of drug-targeting challenges that have faced researchers attempting to treat lysosomal storage diseases by delivering therapeutic enzymes to the lysosome of afflicted cells.