An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes.

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

MicroRNA-mediated regulation of gene expression is affected by disease-associated SNPs within the 3'-UTR via altered RNA structure.

Single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) or their target sites (miR-SNPs) within the 3'-UTR of mRNAs are increasingly thought to play a major role in pathological dysregulation of gene expression. Here, we studied the functional role of miR-SNPs on miRNA-mediated post-transcriptional regulation of gene expression. First, analyses were performed on a SNP located in the miR-155 target site within the 3'-UTR of the Angiotensin II type 1 receptor (AGTR1; rs5186, A > C) mRNA. Second, a SNP in the 3'-UTR of the muscle RAS oncogene homolog (MRAS; rs9818870, C > T) mRNA was studied which is located outside of binding sites of miR-195 and miR-135. Using these SNPs we investigated their effects on local RNA structure, on local structural accessibility and on functional miRNA binding, respectively. Systematic computational RNA folding analyses of the allelic mRNAs in either case predicted significant changes of local RNA structure in the vicinity of the cognate miRNA binding sites. Consistently, experimental in vitro probing of RNA showing differential cleavage patterns and reporter gene-based assays indicated functional differences of miRNA-mediated regulation of the two AGTR1 and MRAS alleles. In conclusion, we describe a novel model explaining the functional influence of 3'-UTR-located SNPs on miRNA-mediated control of gene expression via SNP-related changes of local RNA structure in non-coding regions of mRNA. This concept substantially extends the meaning of disease-related SNPs identified in non protein-coding transcribed sequences within or close to miRNA binding sites.

Noncovalent peptide-mediated delivery of chemically modified steric block oligonucleotides promotes splice correction: quantitative analysis of uptake and biological effect.

Despite numerous encouraging reports in the literature, the efficiency of cell penetrating peptides (CPPs) in promoting cellular delivery of bioactive cargos is still limited. To extend our current understanding of the underlying limitations of such approaches, we performed quantitative uptake studies of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides, targeted against a mutated splice site inserted in a firefly luciferase reporter gene construct, applying the peptide carrier MPGalpha as a model system. The peptide formed stable noncovalent complexes with phosphorothioate oligonucleotide (PTO) and locked nucleic acid (LNA) modified oligonucleotides, whereas the neutral peptide nucleic acid (PNA) had to be hybridized to an unmodified DNA to allow for complex formation. Detailed quantitative uptake studies revealed comparable numbers of intracellular PTO and LNA oligonucleotides after peptide-mediated delivery. Surprisingly, the PTO derivative showed the strongest upregulation of reporter gene activity of about 100-fold followed by the PNA (40-fold) and LNA (10-fold). Electroporation and microinjection studies proved that delivery itself was not the limiting factor for the low activity of the LNA derivative. Maximal achievable reporter gene activity could be observed only after addition of chloroquine (CQ), indicative of an endosomal pathway involved. This is in line with nuclear microinjection experiments, which show that the minimal number of steric block molecules needed to trigger the observed reporter upregulation is about two orders of magnitude lower than determined after peptide or cationic lipid delivery.

Recent developments in peptide-based nucleic acid delivery.

Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect.

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.