Biology subjects

Michalakis, S.

Publications and source records attributed to Michalakis, S..

8 recordsLinked to original sources

bioRxiv2026

Gene Supplementation of MYO7A or activation of Myo7b for treatment of Usher syndrome 1B

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

molecular biology↗

bioRxiv2026

CCZ1 is a modulator of TPC2 activity and melanoma cell migration

The small GTPase RAB7a is a key regulator of melanoma progression by enhancing the activity of the endolysosomal two-pore cation channel TPC2. In this study, we demonstrate that CCZ1--a core component of the RAB7a guanine nucleotide exchange factor (GEF) complex--is essential for mediating this RAB7a-dependent enhancement of TPC2. Unexpectedly, we find that constitutively active (GTP-locked) RAB7a fails to bind and regulate TPC2 in the absence of CCZ1, indicating that CCZ1 contributes to the RAB7a-TPC2 interaction through mechanisms beyond its GEF activity. Furthermore, the CCZ1 facilitated GTPase-activating function on RAB5 is dispensable for modulating TPC2. Notably, in the absence of CCZ1, TPC2 exhibits increased affinity for its agonist, PI(3,5)P2, along with markedly upregulated channel activity. In melanoma cell lines, this upregulation enhances migratory capacity. Our findings identify CCZ1 as a functional inhibitor of TPC2 and highlight its critical role in regulating cancer cell migration.

physiology↗

bioRxiv2025

Crosstalk between the Methyl-Cytosine Dioxygenase TET3 and the Methyl-CpG-binding protein MECP2 Controls Neuronal Maturation

Active DNA demethylation depends on Ten-Eleven-Translocation (TET) enzymes, which oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) and further derivatives. Mutations in TET3, encoding the predominant neuronal isoform, lead to Beck-Fahrner syndrome, a neurodevelopmental disorder. Using human iPSC-derived neurons, we show that TET3 is dispensable for neuronal specification but critical for subsequent maturation. Differentiating TET3-deficient neurons exhibit delayed transcriptional and proteomic transitions, altered synaptic signatures, and impaired network activity, indicating delayed functional maturation. Mechanistically, we identified an interaction between TET3 and the mC/hmC-binding protein MECP2, pathogenic variants of which cause Rett syndrome. MECP2 negatively regulates TET3 activity, as demonstrated in functional assays and by inverse hmC patterns in MECP2- and TET3-deficient neurons. Despite this, MECP2- and TET3-deficient neurons exhibit highly similar phenotypes later in differentiation. Our findings uncover a functional interplay between TET3 and MECP2 that coordinates DNA methylation and chromatin dynamics during neuronal maturation, suggesting a shared pathogenic mechanism in Beck-Fahrner and Rett syndromes.

neuroscience↗

bioRxiv2024

CRISPRa-mediated activation of genes associated with inherited retinal dystrophies in acutely isolated human cells for diagnostic purposes

Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of patients with IRD or other inherited diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/625601v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@60e2b8org.highwire.dtl.DTLVardef@c4c742org.highwire.dtl.DTLVardef@f5c28dorg.highwire.dtl.DTLVardef@b7cf11_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

3D quantification of viral transduction efficiency in living human retinal organoids

The development of therapeutics builds on testing their efficiency in vitro. To optimize gene therapies, for example, fluorescent reporters expressed by treated cells are typically utilized as readouts. Traditionally, their global fluorescence signal has been used as an estimate of transduction efficiency. However, analysis in individual cells within a living 3D tissue remains a challenge. Readout on a single-cell level can be realized via fluorescence-based flow cytometry at the cost of tissue dissociation and loss of spatial information. Complementary, spatial information is accessible via immunofluorescence of fixed samples. Both approaches impede time-dependent studies on the delivery of the vector to the cells. Here, quantitative 3D characterization of viral transduction efficiencies in living retinal organoids is introduced. The approach combines quantified gene delivery efficiency in space and time, leveraging human retinal organoids, engineered adeno-associated virus (AAV) vectors, confocal live imaging, and deep learning-based image segmentation. The integration of these tools in an organoid imaging and analysis pipeline allows quantitative testing of future treatments and other gene delivery methods. It has the potential to guide the development of therapies in biomedical applications.

neuroscience↗

bioRxiv2023

The type of DNA damage response after Decitabine treatment depends on the level of DNMT activity

Decitabine and Azacytidine are considered as epigenetic drugs that induce DNA- methyltransferase (DNMT)-DNA crosslinks, resulting in DNA-hypomethylation and -damage. Although they are applied against myeloid cancers, important aspects of their mode of action remain unknown, which highly limits their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA-damage. Under low DNMT-activity, only Decitabine has a substantial impact. Conversely, when DNMT-activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA-hypomethylation or RNA-associated processes, contradicting an RNA-dependent effect of Azacytidine. By applying spatial proteomics, we show that Decitabine induces a strictly DNMT-dependent multifaceted DNA- damage response based on chromatin-recruitment of various repair-associated proteins. The choice of DNA-repair pathway herby depends on the severity of Decitabine-induced DNA- lesions. While mismatch (MMR) and base-excision DNA repair (BER) as well as RAD50- dependent DNA double-strand break repair are always activated in response to Decitabine, Fanconi anemia-dependent DNA-repair combined with homologous recombination is only activated when DNMT-activity is moderate. In contrast, high DNMT-activity and therefore immense replication stress, induce DNA repair by non-homologous and alternative end-joining.

biochemistry↗

bioRxiv2023

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, intravitreal supplementation of ABCA4 via REVeRT improves retinal degeneration and function in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

bioengineering↗

bioRxiv2022

Human photoreceptor cell transplants integrate into human retina organoids

Cell transplantation is a promising therapeutic approach to recover loss of neurons and vision in patient retinas. So far, human photoreceptor transplants restored some visual function in degenerating mouse retina. Whether retinal cell transplants also integrate into human retina, and how to optimize this for different pathologies are still unknown. Here, we sought to determine if human retina organoids generated from pluripotent stem cells might assist cell replacement therapy development in a human-to-human setting. Models for intra- and subretinal cell transplantation strategies were explored: Photoreceptor donor cells carrying a transgenic fluorescent reporter were enriched from acutely dissociated human retinal organoids. Donor cells were precisely transplanted by microinjection into the retina of host organoids, but high cell numbers might require multiple injections posing potential damage. Alternatively, donor cells were transplanted in large numbers by placing them in subretinal-like contact to the apical organoid surface. Using postmitotic retinal organoids (age >170-days) as a source for donor cells and as hosts, we show that six weeks after subretinal-like transplantation, large clusters of photoreceptors reproducibly incorporate into the host retina. Transplanted clusters frequently are located within or across the host photoreceptor layer, include cone and rod photoreceptors, and become infiltrated by cell processes of host Muller glia, indicative of structural integration. Histological and ultrastructural data of virally-labeled photoreceptor transplants show characteristic morphological and structural features of polarized photoreceptors: inner segments and ribbon synapses, and donor-host cell contacts develop contributing to the retinal outer limiting membrane. These results demonstrate that human retinal organoids provide a preclinical research system for cell replacement therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/500037v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@3a405dorg.highwire.dtl.DTLVardef@57c4deorg.highwire.dtl.DTLVardef@3de9c5org.highwire.dtl.DTLVardef@be9820_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗