Biology subjects

Halbritter, J.

Publications and source records attributed to Halbritter, J..

5 recordsLinked to original sources

bioRxiv2026

Base Editing as a Therapeutic Strategy for Autosomal Dominant Tubulointerstitial Kidney Disease caused by Pathogenic UMOD Variants

Autosomal dominant tubulointerstitial kidney disease caused by pathogenic UMOD variants (ADTKD-UMOD) is an untreatable monogenic kidney disease in which misfolded uromodulin is retained in the endoplasmic reticulum and exerts a toxic gain-of-function effect. Here we systematically evaluate the full spectrum of base editing strategies targeting the UMOD locus. Base editors corrected a representative missense patient variant, p.Cys170Tyr, with A-to-G conversion efficiencies of up to 83.4%, restoring uromodulin trafficking to the plasmamembrane in cell lines and in human 3D kidney tubuloids. Mutation-agnostic inactivation wasachieved by four routes: premature stop codon introduction, splice-site disruption, allele-specific stop codon introduction and promoter attenuation. Maximum on-target efficienciesranged from 44.1% to 71.9%, providing a strategy set applicable across the UMOD allelicspectrum. Endogenous UMOD expression is rarely recapitulated in human cell models,leaving strategies that act on the endogenous gene difficult to assess in patient cells. Wetherefore established CRISPR activation (CRISPRa) of the endogenous UMOD promoterand confirmed its ability to upregulate UMOD expression in human urine-derived cells. Thisapproach further enabled quantification of endogenous UMOD transcript reduction followingbase editing in HEK293 cells. These results establish base editing as a versatile approachfor ADTKD-UMOD and provide a primary human system for testing interventions targetingendogenous UMOD expression, from nucleic acid-based silencing to small molecules.

genetics↗

bioRxiv2026

Cyst-type epithelial heterogeneity shapes therapeutic responsiveness in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) exhibits substantial interpatient variability in disease course and therapeutic response, but the cellular basis for this variability remains poorly understood. Here, we combine single-nucleus RNA sequencing of human cyst epithelia with machine learning-based histological analysis of >1,800 cysts to resolve three epithelial cyst types--proximal tubule-like, collecting duct-like, and mixed. These cyst types display distinct injury states, metabolic programs, and stromal microenvironments, including a mixed-cyst niche enriched for CCL2-associated inflammatory signaling. Expression of key therapeutic targets was highly cell-type specific with CFTR enriched in proximal-like epithelia, whereas AVPR2 expression was confined to AQP2-positive collecting duct-like cells. Cyst-type composition varied widely across patients and in an orthologous mouse model (Pkd1RC/RC) in which the burden of AQP2-positive cysts correlated with responsiveness to tolvaptan. These findings identify cyst-type heterogeneity as a major determinant of molecular pathway activation and predictability of therapeutic response in ADPKD.

molecular biology↗

bioRxiv2025

CAKUT variants in PRPF8, DYRK2, and CEP78: implications for splicing and ciliogenesis

IntroductionCongenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic kidney disease in children and young adults. Although over 50 monogenic causes have been identified, many remain unresolved. PRPF8 is a core spliceosome component, essential for pre-mRNA splicing, and further localizes to the distal mother centriole to promote ciliogenesis. MethodsWe performed trio exome sequencing in 208 CAKUT families and identified strong variants in PRPF8 and the EDD-DYRK2-DDB1VprBP complex. Functional validation included splicing assays in yeast (Saccharomyces cerevisiae), Sonic hedgehog (Shh) signaling in RPE-1 cells, co-immunoprecipitation for protein complex assembly, and in situ hybridization in mouse embryos. Protein interactions were modeled using AlphaFold. ResultsWe identified heterozygous de novo or inherited variants in PRPF8, DYRK2, DDB1, EDD and CEP78. Yeast assays revealed that while most PRPF8 variants preserved growth and splicing at consensus splice sites, the de novo PRPF8R1681W variant impaired splicing of non-consensus splice sites and was inviable at elevated temperature. CAKUT variants failed to rescue prp28-1 and U4-cs1 alleles but showed variant-specific synthetic interactions with brr2-1, including weak suppression or synthetic sickness at elevated temperatures. Shh signaling was reduced in [~]50% of PRPF8 variants expressed in RPE-1 cells. CEP78 truncating variants abrogated binding to CEP350 and VPRBP. Two DYRK2 variants disrupted EDD-DYRK2-DDB1VprBP complex formation without affecting kinase activity. In situ hybridization revealed strong Prpf8 expression in the developing collecting duct and urothelium. ConclusionVariants in PRPF8 and components of the EDD-DYRK2-DDB1VprBP complex may contribute to CAKUT through impaired pre-mRNA splicing and defective ciliogenesis. These findings uncover an entirely new functional network of candidate genes for CAKUT and ciliopathies, significantly broadening our understanding of disease mechanisms and offering novel entry points for mechanistic studies. Translational StatementOur study identifies a previously unrecognized molecular network involving PRPF8 and the EDD-DYRK2-DDB1VprBP complex, revealing a novel pathogenic mechanism in CAKUT. These results introduce a new class of candidate genes and pathways essential for kidney development. As the genetic etiology of CAKUT remains unknown in most patients, our findings underscore the need for targeted genetic testing and functional studies to enhance diagnosis, advance mechanistic insight, and enable more personalized clinical management.

genetics↗

bioRxiv2025

In vivo base editing reduces liver cysts in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants in PKD1 or PKD2 account for 85-90% of cases. Genetic re-expression of Pkd1 or Pkd2 has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in the understanding of the genetic basis, it remains unclear whether the correction of underlying pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of extrinsically delivered genome editing as a treatment option for ADPKD remains largely unexplored. In this study, we employed CRISPR base editing to correct a spectrum of representative pathogenic PKD1 variants selected from a patient cohort achieving precise and efficient editing in vitro. Correction of a representative murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells successfully increased polycystin-1 expression and reduced levels of the endoplasmic reticulum stress marker sXBP1. In vivo, base editor delivery to the c.6646C>T (R2216W) knock-in mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence of ADPKD reversibility through genome editing, opening promising novel therapeutic perspectives for affected patients and their families.

genetics↗

bioRxiv2019

A deregulated stress response underlies distinct INF2 associated disease profiles

Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Here we systematically characterize a large panel (>50) of autosomal dominant mutants of the actin regulator inverted formin 2 (INF2) that have been reported to cause the podocytic kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot Marie-Tooth disease (CMT). We found that INF2 mutations lead to deregulated activation of the formin and a constitutive stress response in cultured cells, primary patient cells and Drosophila nephrocytes. Using quantitative live-cell imaging we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. Furthermore, we could clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and CMT. Our results indicate that cellular profiling of disease-associated mutations can contribute substantially to sequence-based phenotype predictions.

cell biology↗