Biology subjects

Nagiel, A.

Publications and source records attributed to Nagiel, A..

5 recordsLinked to original sources

bioRxiv2026

Allele-specific correction of dominant Best vitelliform macular dystrophy in patient-derived retinal pigment epithelium

Autosomal dominant Best vitelliform macular dystrophy (BVMD) caused by variants in the BEST1 gene is characterized by dysfunction of the macular retinal pigment epithelium (RPE) and secondary degeneration of the photoreceptors. There are currently no approved treatments for BVMD, and owing to its dominant nature, there remains uncertainty regarding the utility of traditional gene augmentation. Here we evaluated whether a dominant pathogenic BEST1 allele can be corrected by base editing in differentiated RPE cells. We identified a patient with a likely pathogenic BEST1 c.851A>G (p. Tyr284Cys) variant that was amenable to cytidine base editing. After establishing patient-derived induced pluripotent stem cells (iPSCs), we corrected the pathogenic variant in the iPSCs to obtain corrected iPSCs with the same genetic background. Corrected iPSC-derived RPE exhibited normalized monolayer appearance, improved barrier integrity, reduced cell death, and restored RPE-specific transcriptome. We then used a dual adeno-associated virus (AAV) split-intein system to deliver a CRISPR-associated protein 9 cytidine base editor (SpCas9-CBE) to BEST1 c.851A>G mutant RPE monolayers and achieved editing of the pathogenic allele with a maximum efficiency of 13.42 {+/-} 3.64% (mean {+/-} SD). Together, these results demonstrate progress towards allele-specific base editing in a dominantly inherited retinal disorder.

neuroscience↗

bioRxiv2026

mGluR6 coordinates cone terminal targeting and synaptic layer assembly during human retinal development

The metabotropic glutamate receptor 6 (mGluR6), encoded by GRM6, is a core component of the ON-bipolar signaling cascade in the retina, but its role in human retinal development remains unclear. Here, we used temporally controlled CRISPR-based genetic ablation in human induced pluripotent stem cell-derived retinal organoids to define the developmental functions of mGluR6. Unexpectedly, we found that mGluR6 is expressed not only in depolarizing ON-bipolar cells but also transiently in cone photoreceptors during human retinal development, a pattern not observed in the mouse retina. Early loss of GRM6 prior to synaptogenesis disrupted cone pedicle architecture, leading to mislocalization of synaptic proteins including Bassoon, ELFN2, and TRPM1, and ultimately resulting in widening or duplication of the outer plexiform layer (OPL). In contrast, deletion after synapse formation did not alter OPL synapses or morphology, revealing a temporally restricted requirement for mGluR6 during circuit assembly. These findings uncover a previously unrecognized role for mGluR6 in coordinating cone terminal targeting and synaptic layer assembly during human retinal development and highlight the power of temporally controlled genetic manipulation in organoid systems to reveal species-specific mechanisms of neural circuit formation.

neuroscience↗

bioRxiv2022

Non-Synonymous, Synonymous, and Non-Coding Nucleotide Variants Contribute to Recurrently Altered Biological Processes During Retinoblastoma Progression

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To assess synonymous and non-coding variant contributions to recurrently altered processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to initiating RB1 mutations, MYCN amplification, and established retinoblastoma SCNAs, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, inclusion of non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we performed functional tests of 3 UTR variants of PCGF3 (a BCOR-binding component of Polycomb repressive complex I) and CDC14B (a regulator of sister chromatid segregation) and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). PCGF3 and CDC14B 3 UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protein structure and stability. Compared to tumors, retinoblastoma cell lines had a partially overlapping variant gene spectrum and enrichment for p53 pathway mutations. These findings reveal potentially important differences in retinoblastoma cell lines and antecedent tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

cancer biology↗

bioRxiv2022

Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular stress can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily post-mitotic and aids regeneration in mammals. In the present study we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of its target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies. Significance StatementIn humans and other mammals, many organs regenerate through the proliferation of cells that replace those that have succumbed to aging or injury. However, proliferation is largely absent in certain critical organs, including the heart, the central nervous system, and sensory organs such as the inner ear and retina. The Hippo-Yap biochemical signaling pathway, a cascade of proteins that--when active--inhibits cell division, constitutes one impediment to proliferation. We earlier identified a small molecule that interrupts Hippo-Yap signaling and thus relieves this block for some non-proliferating cells in vitro. In the present investigation, we have chemically modified the original substance to yield a more potent analog that is effective for several hours in mammalian tissues in vivo and initiates the proliferation of heart-muscle cells after cryolesioning. After further refinements, compounds of this family might prove useful in regenerative therapies.

cell biology↗

bioRxiv2020

Small-molecule inhibition of Lats kinases promotes Yap-dependent proliferation in postmitotic mammalian tissues

Hippo signaling is an evolutionarily conserved pathway that restricts organ growth during development and suppresses regeneration in mature organs1-3. Using a high-throughput phenotypic screen, we have identified a potent, non-toxic, and reversible inhibitor of Hippo signaling. An ATP-competitive inhibitor of Lats kinases, the compound causes Yap-dependent proliferation of murine supporting cells in the inner ear, murine cardiomyocytes, and human Muller glia in retinal organoids. RNA sequencing indicates that the substance fosters both the G1-S and G2-M checkpoint transitions and yields supporting cells capable of transdifferentiation. Upon withdrawal of the compound, a subset of supporting cells move their nuclei into the hair-cell layer and express genes characteristic of hair cells. Viral transfection of Atoh1 induces the expression of hair cellspecific proteins in progeny. The compound promotes the initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

developmental biology↗