Andrea Rossi

Mitochondrial DNA (mtDNA) variants, whether maternally inherited or acquired somatically during aging, can be particularly detrimental to cells that rely on mitochondrial metabolism such as neurons. The functional consequences of mtDNA variants are thought to depend on their heteroplasmy, defined as the proportion of mtDNA molecules carrying that variant. However, investigating the specific effects of heteroplasmic mtDNA variants on human neuronal function has been challenging. Studies using patient-derived cells cannot conclusively attribute observed phenotypes to a single mtDNA variant, while conventional genome editing approaches are not applicable to mtDNA. Here, we use DddA-derived cytosine base editor (DdCBE) to introduce pathogenic heteroplasmic mtDNA variants into Neurogenin 2 (NGN2)-inducible human induced pluripotent stem cells (iPSCs). We focus on two variants in the genes MT-ND1 and MT-ND5 belonging to complex I (CI), the largest component of the mitochondrial respiratory chain and a frequent contributor to human disease, including the incurable neurodevelopmental disorder Leigh syndrome. mtDNA-edited iPSC lines exhibit low off-target editing and maintain similar heteroplasmy following subclonal isolation and differentiation into NGN2-induced neurons (iNs). Although the CI variants do not substantially affect mitochondrial energy metabolism in undifferentiated iPSCs, they reduce mitochondrial membrane potential in iNs where they drive a transcriptional reprogramming indicative of impaired neuronal commitment. These findings suggest that heteroplasmic CI variants are sufficient to disrupt neural fate specification even at relatively low heteroplasmy. More broadly, mtDNA editing in human iPSCs provides a powerful platform to investigate how mitochondrial fitness and heteroplasmy influence development and disease.

Heterozygous loss-of-function (LoF) variants are pervasive in human genomes, yet the mechanisms that preserve gene dosage remain poorly understood. Analysis of phased GTEx transcriptomes revealed widespread donor-specific allele depletion, including in dosage- sensitive genes, whereas haplotype-resolved transcriptomes from the Human Pangenome Reference Consortium identified naturally occurring pLoF-associated patterns consistent with preserved total expression alongside greater relative contribution from the intact haplotype. Using isogenic human iPSC models carrying complementary heterozygous disruptions of ACTB, we define allele-specific transcriptional rebalancing (ASTR) as a compensatory increase in productive RNA output from the intact allele. Despite near-complete loss of mutant-allele output, ASTR maintained ACTB RNA and protein and emerged during RNA maturation rather than transcriptional initiation, without global transcriptional amplification. Haplotype-resolved Fiber-seq further suggested mutation-class-specific chromatin configurations accompanying rebalancing. Similar rebalancing following heterozygous disruption of the unrelated dosage-sensitive gene POU5F1 showed that ASTR is not restricted to ACTB. Single-cell transcriptomics revealed convergent depletion of cellular states linked to stress resilience, including metal-ion response and detoxification, following independent ACTB and POU5F1 disruption. Direct cadmium challenge revealed increased sensitivity in compensated cells, and broader environmental perturbation profiling showed altered transcriptional stress responses despite preserved baseline dosage. Together, these findings identify ASTR as a gene-dosage-buffering response in human cells and show that preservation of molecular output can coexist with reduced adaptive reserve.

Mitochondrial diseases are highly complex and heterogeneous, and nearly 20% of cases involve severe liver pathology. Here, we report an affected individual carrying a pathogenic MIC13 variant (c.260-2 A > G) associated with early-onset mitochondrial hepato-encephalopathy. Such mitochondrial hepatopathies are rare, multisystemic disorders with major liver involvement, difficult to diagnose, lack effective treatment, and are poorly understood, in part due to the absence of faithful disease-relevant cellular models. To investigate hepatocyte-specific consequences of the MIC13 variant, we generated iPSCs carrying this disease-causing variant and differentiated them into induced hepatocytes (iHeps). MIC13, a key component of the MICOS complex required for cristae formation, was disrupted in these cells, and the resultant iHeps exhibited the same cristae defects observed in clinical samples. Integrated multi-omics and biochemical analyses revealed extensive metabolic rewiring, including disrupted amino acid turnover, accumulation of tricarboxylic acid (TCA) and urea cycle intermediates. Additionally, profound alterations in methionine cycle and transsulfuration pathways, along with enhanced bile acid synthesis, collectively affect methylation potential, redox homeostasis, and detoxification. Lipid metabolism was also impaired, with incomplete β-oxidation, increased ketogenesis, and diminished lipid storage. At the cellular level, extensive extracellular matrix (ECM) remodeling, increased intracellular collagen accumulation and enhanced cell migration indicated an early fibrotic phenotype, linking metabolic rewiring to ECM homeostasis. Overall, this clinically relevant model uncovers how cristae defects drive metabolic imbalance and hepatocyte dysfunction, ultimately leading to early fibrotic changes in mitochondrial liver disease. These findings provide a strong mechanistic foundation for understanding mitochondrial liver disease and developing targeted therapeutic strategies.

Mitochondriopathies are a large group of pathological conditions characterized by dysfunction of tissues and organs with high energy needs such as the brain, heart, and skeletal muscles, which highly rely on functional mitochondrial oxidative phosphorylation (OXPHOS). The vast majority of these disorders are ascribed to mutations in nuclear or mitochondrial encoded subunits of complex I (CI), the largest OXPHOS complex. In this study, we develop a multisystemic pipeline for CI-associated disease based on new approach methods (NAMs), which includes non-mammalian models, namely C. elegans and zebrafish, and mammalian cell-based models, such as neuroblastoma cells and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) and brain organoids. Systematic characterization of biochemical and neurobehavioral features in these models reveals obvious mitochondrial alterations with no major signs of redox imbalance possibly due compensatory or tissue-specific effects. Conversely, we identify specific pathogenetic features that are consistent across species and are rescued by lutein or vitamin B12, likely acting through non-cell-autonomous mechanisms converging on neurometabolic rewiring. Overall, our findings support the advantage of leveraging different in vitro and in vivo NAM systems with their unique and complementary disease endpoints to uncover critical pathological and quantifiable phenotypes exploitable to identify disease modifiers and suppressors.

Loss of filaggrin (FLG) function impairs skin barrier formation and contributes to common inflammatory skin diseases. In this study, we established a FLG knockout human iPSC line based on KOLF2.1J using CRISPR/Cas12a-mediated genome editing. A guide RNA targeting exon 2 introduced a homozygous mutation, which was confirmed by sequencing. The edited cells maintained typical pluripotent stem cell morphology, expressed key undifferentiated markers, and retained the ability to differentiate into all three germ layers. Karyotype and CNV analyses confirmed genomic stability and parental origin; the cells were free of mycoplasma. This cell line enables studies of FLG-associated skin biology and pathology.