ZIP12, a membrane-bound zinc importer, is up-regulated in the remodeled pulmonary arterioles of patients with pulmonary arterial hypertension (PAH). Genetic deletion of its encoding gene, Slc39a12 , ameliorates the disease in rodent models. Here, we show that ZIP12 overexpression in human pulmonary artery smooth muscle cells (PASMCs) increased intracellular labile Zn 2+ and promoted proliferation. ZIP12 siRNA knockdown attenuated pulmonary hypertension in rodent models. We developed a humanized anti-ZIP12 monoclonal antibody, APL-9796, that normalized intracellular Zn 2+ concentration, suppressed proliferation in fibroblasts derived from patients with PAH and PDGF-stimulated PASMCs, and reduced PDGF-induced cytoskeletal remodeling in PASMCs and contractility in PASMCs and engineered pulmonary artery tissues. APL-9796 showed dose-dependent efficacy in humanized Slc39a12 rats, improving cardiopulmonary parameters after induction of pulmonary hypertension and was well tolerated in phase 1 clinical studies. Transcriptomic profiling revealed modulation of several molecular pathways, including those involving AMPK, PPAR, and PI3K-Akt. ZIP12-driven labile Zn 2+ signaling underpins the pathogenesis of PAH. Targeting ZIP12 with APL-9796 offers a promising therapeutic strategy.
Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease, the most common form of autosomal dominant cerebellar ataxia, is caused by a CAG repeat expansion in ATXN3 . The mutant protein is prone to aggregation and exerts toxic gain-of-function effects, whereas the wild-type protein has important neuroprotective roles as a deubiquitinase. Current treatment strategies for SCA3 lack specificity for the mutant allele. Identifying genetic variants such as single-nucleotide polymorphisms (SNPs) in linkage with the pathogenic CAG expansion offers a promising avenue to develop allele-specific therapeutic interventions, like antisense oligonucleotides (ASOs). To address this, we conducted allele-specific, targeted, multiplexed nanopore sequencing on 79 patients with SCA3 from Germany, UK, and Portugal. We identified almost 200 polymorphisms and grouped them into three haplotype blocks on the basis of linkage disequilibrium. These SNPs were strongly associated with the CAG expansion. Genotypic modeling of our subcohorts indicated that targeting only two SNPs from different blocks could enable allele-specific treatment of the majority of European patients with SCA3. We evaluated 17 candidate ASOs targeting SNPs from different haplotype blocks. Seven of these ASOs caused allele-preferential reduction of more than 65%. A single ASO application in two SCA3 iPSC-derived cortical neurons (AX1, AX2) achieved up to 90% reduction of mutant ATXN3. Toxicity assessments together with RNA sequencing analyses in AX1 did not indicate evidence of toxicity or widespread transcriptional perturbations. These findings highlight that characterizing heterozygosity rates offers valuable opportunities for selective ASO therapies. Our proof-of-principle targeting with ASOs demonstrates the feasibility of haplotype-informed allele-specific silencing in ATXN3 .
Amphotericin B remains the gold-standard therapy for invasive fungal infections, but its clinical utility is limited by toxicity arising from poor selectivity for fungal ergosterol over mammalian cholesterol, a challenge that decades of medicinal chemistry have failed to fully overcome. Here, we report mandimycin M16, a polyene macrolide engineered from a unique 5 + 1 polyene scaffold that uncouples antifungal potency from host toxicity. Mandimycin M16 exhibited potent, broad-spectrum activity against 11 tested World Health Organization (WHO) priority fungal pathogens. Unlike amphotericin B, which binds cholesterol, M16 exhibited no detectable cholesterol binding and showed no detectable hemolysis, cytotoxicity, or nephrotoxicity at concentrations up to 128 micrograms per milliliter in vitro and after up to seven daily intravenous doses in mice. Mechanistic studies revealed that mandimycin M16 exerted rapid fungicidal activity through selective recognition of the conjugated C5–C8 diene system present in ergosterol but absent in cholesterol, a mode of sterol discrimination fundamentally different from classical polyenes. In multiple disseminated murine infection models, M16 outperformed liposomal amphotericin B at equivalent doses, achieving up to 3.4 log reduction in fungal burden and 100% survival without detectable renal injury by biomarkers or histopathology. These findings establish that the 5 + 1 polyene architecture enables complete sterol selectivity and molecularly uncouples antifungal potency from host toxicity, providing a next-generation antifungal candidate for the treatment of invasive fungal infections.
Expanded CAG/CTG repeats cause more than 15 different diseases that all remain without a disease-modifying treatment. Because repeat length accounts for most of the variation in disease severity, contracting CAG/CTG repeats presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeted to CAG/CTG repeats leads to efficient repeat contractions in Huntington’s disease (HD) patient–derived neurons and astrocytes and in myotonic dystrophy type 1 patient–derived neurons. The approach was allele selective and free of detectable off-target mutations. Injection of the Cas9 nickase into the striatum of the R6/1 mouse model of HD using adeno-associated viral vectors led to repeat contractions in more than half of striatal neurons. Upon injection, we observed a reduction in the number of inclusion bodies, an improved gene expression profile, and amelioration of locomotion deficits in the open field test. The effects were greater than expected from the contractions induced, suggesting that non–cell autonomous mechanisms may be involved. Our results provide the proof of concept that correction of CAG/CTG repeats can improve HD phenotypes in a mouse disease model.
Cerebral cavernous malformations (CCMs) constitute the second most common type of intracranial vascular malformations. Cavernomas arise because of somatic or autosomal dominant inherited loss-of-function mutations in CCM genes, forming fragile mulberry-like malformed blood vessel structures that are prone to leak and break. Treatment is limited to surgical removal of solitary cavernomas, and because patients often present with multiple lesions, pharmacological alternatives to surgery are needed. We performed a high-throughput screen of 5200 bioactive molecules in programmed cell death 10 (PDCD10 or CCM3)–deficient mouse endothelial cells, coupled to automated image analysis. This screen identified dabrafenib, a clinically approved BRAF inhibitor, as a compound that restored cell-cell junctions and vascular barrier properties of CCM3-deficient endothelial cells. The potential translatability of dabrafenib was tested in acute and chronic CCM mouse models, where it reduced cerebral lesion burden and suppressed lesion permeability. Dabrafenib also rescued abnormal structural defects present in the developing retina vasculature in an acute CCM mouse model. Mechanistically, dabrafenib targeted extracellular regulated kinase (ERK)5–Krüppel-like factor (KLF)2/4 signaling and suppressed F-actin stress fiber formation in human brain endothelial cells, two potentially independent and central molecular processes involved in CCM pathology. Silencing BRAF did not prevent dabrafenib-induced KLF2/4 inhibition or restoration of cell-cell junctions, suggesting that its dual therapeutic outcome is independent of BRAF pathway inhibition. In conclusion, dabrafenib is a low–molecular weight compound that inhibits cavernoma pathogenesis, suggesting that CCMs could be treated with dabrafenib to block cavernoma growth and vascular leakage.
