Biology subjects

Borsos, M.

Publications and source records attributed to Borsos, M..

7 recordsLinked to original sources

bioRxiv2026

AAV-DJ Enables Targeted Gene Modulation in Human Ovarian Cells

Study question: Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation? Summary answer: AAVs can efficiently transduce human primary granulosa cells and human ovarian stromal cells with low toxicity, with the serotype DJ (AAV-DJ) demonstrating superior performance. AAVs enable targeted modulation of ovarian genes such as AMHR2 in human primary granulosa cells and TGF{beta}1 in human ovarian stromal cells, supporting the feasibility of gene-based approaches to improve ovarian function. What is known already: Infertility affects approximately 15% of couples, with diminished oocyte quality and quantity being major contributors, particularly with advanced reproductive age. Assisted reproductive technologies rely primarily on hormonal stimulation and do not directly target molecular pathways governing folliculogenesis or the ovarian microenvironment. Animal studies suggest that gene therapy using AAVs offers a promising strategy for tissue-specific and durable gene modulation, yet its application in human ovarian cells remains largely unexplored. Study design, size, duration: This was an experimental laboratory study using primary human granulosa cells from patients undergoing in vitro fertilization (IVF) and ovarian stromal cells obtained from post-pubertal patients who donated surplus ovarian tissue from ovarian tissue cryopreservation procedures for research. Sixteen AAV serotypes were evaluated for transduction efficiency, toxicity, and expression dynamics, followed by functional gene knockdown studies using AAV-DJ-based shRNA vectors. Participants/materials, setting, methods: Primary human granulosa cells and ovarian stromal cells were isolated from follicular aspirates and ovarian tissue, respectively. Cells were transduced with GFP-expressing AAV serotypes at varying multiplicities of infection (MOIs), incubation times, and culture days to assess transduction efficiency and cytotoxicity. Lead serotypes were further evaluated in dose-response studies. Functional gene modulation was assessed using AAV-DJ-mediated shRNA knockdown of AMHR2 in granulosa cells and TGF{beta}1 in stromal cells, quantified by RT-qPCR, immunofluorescence, and western blotting. Main results and the role of chance: Six of sixteen AAV serotypes (AAV2, AAV3, AAV6, AAV9, AAV-DJ, and AAV-X1.1) achieved greater than 10% transduction efficiency in human primary granulosa cells. Among these, AAV-DJ demonstrated the highest efficiency across all MOIs tested, reaching up to 48% transduction at 100,000 MOI, significantly outperforming AAV2 and AAV9. Toxicity increased with viral dose for all serotypes but remained comparable across vectors, with AAV-DJ exhibiting higher efficiency with comparable toxicity. The AAV-DJ-shRNA-AMHR2 construct produced a significant reduction in expression of AMHR2 mRNA by ~81% (p<0.05) in human primary granulosa cells as well as at the protein level in HEK293T-AMHR2-ZsGreen engineered cells (~85% knockdown, p<0.01). In human ovarian stromal cells, AAV-DJ preferentially transduced EMILIN1 and ACTA2-positive fibroblast and myofibroblast-like cells and enabled robust knockdown of TGF{beta}1 at both transcript (~89% knockdown, p<0.05) and protein levels (~67% knockdown, p<0.0001) using AAV-DJ-shRNA-TGF{beta}1 construct. Limitations, reasons for caution: These experiments were performed in vitro using primary human ovarian cells, which may not fully recapitulate the in vivo ovarian environment. Functional reproductive outcomes were not assessed, and future studies will be required to evaluate the effects of gene modulation on folliculogenesis and fertility. Wider implications of the findings: These findings establish AAV-DJ as a promising vector for targeted gene modulation in human ovarian cells. This approach introduces a novel framework for addressing infertility and ovarian function by directly targeting molecular pathways involved in follicle recruitment and ovarian stromal signaling, potentially enhancing current assisted reproductive technologies.

developmental biology↗

bioRxiv2026

Defining a Midgestational Window for In Utero Genome Editing of the Fetal Murine Cortex

Congenital disorders of cortical development arise from genetic lesions that disrupt neurogenesis and neuronal migration. Unfortunately, tools to model or correct these defects before birth are limited. Here we establish a platform for systemic in utero gene delivery and genome editing in the mouse cortex at midgestation. By microdissecting a uterine window over the vitelline vein at embryonic day 12.5 (E12.5), we achieve fetal circulation access, enabling robust AAV-mediated transduction of the central nervous system (CNS) while reducing off-target expression in peripheral organs. Barcoded capsid screens reveal that AAV9 exhibits developmental stage-dependent tropism, with higher CNS penetrance and lower liver transduction at E12.5 than at E15.5. Leveraging this window, we provide a proof-of-concept of efficient cortical editing, using Cre-lox and CRISPR/Cas9 strategies to recapitulate prenatal reeler-like cortical misordering phenotypes following Reln knockout. We further use homology-directed repair to demonstrate precise genome modification, epitope-tagging the endogenous Reln and Actb loci, and installing a human-derived pathogenic allele of PDHA1. Importantly, we show that edited cells span neural progenitors and differentiated neurons across the cortex and hippocampus. These results define a permissive midgestational window for prenatal genome editing, providing a platform for functional modeling of congenital CNS disorders and exploration of early therapeutic interventions with minimized peripheral exposure.

neuroscience↗

bioRxiv2023

Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes. HighlightsO_LITranscriptional crosstalk between enhancers and promoters delivered in trans by AAVs is a generalized phenomenon. C_LIO_LISpatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk. C_LIO_LITranscriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation. C_LIO_LITranscriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts. C_LI

bioengineering↗

bioRxiv2023

Dysregulated mammalian estrus cycle rescued by timed activation of VIP neurons in the circadian pacemaker and late afternoon light exposure

Jet lag and shift work disrupt the menstrual cycle and decrease fertility. The circadian pacemaker, the suprachiasmatic nucleus (SCN), is known to modulate ovulation, but the mechanism is unclear. Here we explore this connection by tracking the dynamics of vasoactive intestinal peptide (VIP)-expressing neurons in the SCN in freely-behaving mice. We show that SCNVIP activity is time-of-day- and sex-dependent, and estrous-state-dependent in late afternoon, gating downstream activation of GnRH neurons. Afternoon light, as well as specific activation of SCNVIP neurons, rescues estrous cycle regularity and egg release in animals in altered light conditions, emphasizing the role of SCNVIP neurons as a time-dependent light-responsive switch. Our results reveal the dynamic mechanism by which SCNVIP neurons mediate light responses to regulate estrous states and demonstrate light-induced fertility rescue. One Sentence SummaryModulating and recording the activity of suprachiasmatic VIP neurons in freely behaving mice reveals their regulation of fertility by mediating the response to late afternoon light.

neuroscience↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗

bioRxiv2020

Mechanism of cell polarisation and first lineage segregation in the human embryo

The formation of differential cell lineages in the mammalian blastocyst from the totipotent zygote is crucial for implantation and the success of the whole pregnancy. The first lineage segregation generates the polarised trophectoderm (TE) tissue, which forms the placenta, and the apolar inner cell mass (ICM), which mainly gives rise to all foetal tissues and also the yolk sac1-3. The mechanism underlying this cell fate segregation has been extensively studied in the mouse embryo4,5. However, when and how it takes place in the human embryo remains unclear. Here, using time-lapse imaging and 325 surplus human embryos, we provide a detailed characterisation of morphological events and transcription factor expression and localisation to understand how they lead to the first lineage segregation in human embryogenesis. We show that the first lineage segregation of the human embryo is triggered by cell polarisation that occurs at the 8-cell stage in two sequential steps. In the first step, F-actin becomes apically polarised concomitantly with embryo compaction. In the second step, the Par complex becomes polarised to form the apical cellular domain. Mechanistically, we show that activation of Phospholipase C (PLC) triggers actin polarisation and is therefore essential for apical domain formation, as is the case in mouse embryos6. Finally, we show that, in contrast to the mouse embryo, the key extra-embryonic determinant GATA37,8 is expressed not only in extra-embryonic lineage precursors upon blastocyst formation. However, the cell polarity machinery enhances the expression and nuclear accumulation of GATA3. In summary, our results demonstrate for the first time that cell polarisation reinforces the first lineage segregation in the human embryo.

developmental biology↗

bioRxiv2019

GPIF constrains Polycomb Repressive Complex 2 activity in germ cells

The Polycomb machinery is required for the proper orchestration of gene expression by virtue of its critical role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a new cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This alteration of the epigenetic content of mature oocytes does not affect the initial number of follicles but is associated with a reduction of follicles in aging mice. We provide evidences that mature oocytes Ezhip -/- are not fully functional and that fertility is strongly impaired in Ezhip -/- females. Altogether, our study uncovers EZHIP as a novel functional player in the comprehensive chromatin remodeling that occurs in the gonads.

developmental biology↗