Biology subjects

Mandai, M.

Publications and source records attributed to Mandai, M..

13 recordsLinked to original sources

bioRxiv2026

Mutation-agnostic gene insertion therapy for RHO-associated autosomal dominant retinitis pigmentosa using zinc finger nucleases

PurposeAutosomal dominant retinitis pigmentosa caused by mutations in the rhodopsin gene (RHO-adRP) is among the most prevalent inherited retinal dystrophies. With nearly 100 distinct pathogenic variants identified to date, the mutational heterogeneity of RHO-adRP severely limits the clinical utility of mutation-specific therapeutic strategies. We aimed to develop a mutation-agnostic gene insertion therapy using homology-independent targeted integration (HITI) mediated by zinc finger nuclease ZF-ND1 targeting the human RHO 5'-UTR, and to validate its preclinical efficacy, safety, proof-of-concept, and proof-of-mechanism. MethodsWe developed two AAV serotype 5 (AAV5) vectors: one encoding the ZF-ND1 pair (AAV5-ZFN) and one carrying the therapeutic donor cassette (AAV5-RHO), delivered by subretinal co-injection. ZF pairs targeting the RHO 5'-UTR were arranged and refined by in vitro validation; AAV vector optimization and mechanistic verification were performed in human induced pluripotent stem cell (hiPSC)-derived retinal organoids-derived retinal organoids; longitudinal proof-of-concept efficacy and safety were assessed in a humanized RHO-T17M rat disease model by 6-month optical coherence tomography (OCT); and proof-of-mechanism was evaluated in non-human primate retina. ResultsWe identified a ZF-ND1 pair achieving cleavage efficiency comparable to the SpCas9 RNP previously validated for HITI-mediated editing in mouse retina, and optimized the ZF array composition and NLS configuration for efficient editing especially in post-mitotic photoreceptors. HITI-mediated donor integration was confirmed across multiple cell types and ZFN:donor ratios. In the humanized rat disease model, the therapeutic vector provided outer nuclear layer (ONL) preservation by 6 months, with AAV5-ZFN:AAV5-RHO ratios of 1:1 and 1:2 maintaining ONL thickness above the preservation threshold. In the non-human primate retina, the fraction of HITI-edited rod photoreceptors exceeded the 20% therapeutic correction threshold in the successfully treated individual. ConclusionsThese findings support the advancement of this therapeutic vector to first-in-human trials as a mutation-agnostic insertion therapy applicable to all patients with RHO-adRP, irrespective of the specific causative variant.

genetics↗

bioRxiv2026

Plagl2 unlocks the latent regenerative potential of Müller glia in the adult mouse retina

Muller glia (MG) in the adult mammalian retina have long been recognized as a potential endogenous source for the regeneration of lost retinal neurons. However, existing MG reprogramming strategies yield an incomplete regenerative response by favoring either proliferation or neurogenic differentiation. Here, we show that the zinc-finger transcription factor Plagl2, which has been demonstrated to rejuvenate aged neural stem cells, was sufficient to reprogram adult mouse MG into a progenitor-like state with coupled proliferative and neurogenic competence. Using histology, time-lapse imaging, and single-cell transcriptomics, we found that Plagl2 drove regulated rounds of MG cell cycle re-entry, while N-methyl-D-aspartate-induced retinal injury further promoted the acquisition of neurogenic competence toward inner nuclear layer neuronal identities. These findings identify Plagl2 as a novel rejuvenator of mammalian MG and support the general principle that reprogramming modules can be redeployed across cell types, offering new avenues for unlocking regenerative potential in otherwise non-regenerative tissues.

neuroscience↗

bioRxiv2025

Chemotherapy Induces an IL1β-dependent Neutrophil Recruitment that Promotes Chemoresistance in Metastatic Ovarian Cancer

High-grade serous carcinoma (HGSC) of the ovary acquires chemoresistance through diverse cancer cell-intrinsic and-extrinsic mechanisms, culminating in treatment-refractory intraperitoneal metastasis. How chemotherapy-induced remodeling of the tumor microenvironment modulates drug sensitivity remains unclear. In this study, we demonstrate that chemotherapy induced IL1{beta}-dependent neutrophil accumulation in tumors, driving chemoresistance in HGSC. Using patient samples, bulk transcriptomic profiling before and after chemotherapy revealed post-treatment upregulation of IL1B, and single-cell RNA sequencing identified myeloid cells as its principal source. In a chemoresistant murine metastatic ovarian cancer model, chemotherapy increased neutrophils and neutrophil extracellular traps (NETs) in omentum tumors; these increases were abrogated in IL1{beta}-deficient mice, with expansion of activated CD8+ T cells and tumor control. Neutrophil depletion in wild-type mice recapitulated the chemosensitive phenotype of IL1{beta}-deficient mice. In vitro, IL1{beta} did not alter cancer cell-intrinsic chemosensitivity, whereas NETs reduced the chemosensitivity of cancer cells. Additionally, the dominant IL1{beta} receptor (IL1R1) was predominantly expressed in tumor-associated fibroblasts in humans and mice. Consistently, IL1R1-deficient mice exhibited chemosensitivity with decreased neutrophil accumulation and increased IFN{gamma}TNFCD8 T cells. We also found that chemotherapy upregulated CXCL2 in patients and that ablating IL1{beta}-IL1R1 axis decreased CXCL2 expression in tumor-associated fibroblasts in mice. Finally, residual human HGSC tumor after chemotherapy showed increased neutrophils and a trend toward more NETs. Collectively, these findings illuminate a paradoxical, cancer cell-extrinsic mechanism in HGSC whereby chemotherapy itself amplifies chemoresistance and suggest that targeting chemotherapy-induced inflammation may help overcome treatment resistance.

immunology↗

bioRxiv2025

Genome-edited retinal organoids restore host bipolar connectivity in the primate macula

Retinal organoids (ROs) represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but whether host cone bipolar cells (BCs) in the primate macula can rewire with transplanted photoreceptors remains unresolved. Here, we transplanted genome-edited human retinal organoids lacking ON-BCs (Islet-1-/- ROs) into a non-human primate macular degeneration model. Remarkably, host rod and cone BCs extended dendrites toward grafted photoreceptors, forming functional synapses confirmed by immunohistochemistry, ultrastructural imaging, and focal macular electroretinography. Both ON- and OFF-pathway connectivity was rebuilt, providing the first demonstration of host-graft synaptic integration in the primate macula. These results establish that primate cone circuits retain a surprising capacity for rewiring and highlight genome-edited ROs as a powerful platform for vision restoration. Our findings represent a critical translational step toward stem cell-based therapies capable of repairing central vision in patients with advanced macular degeneration.

neuroscience↗

bioRxiv2025

Hidden Markov Models Reveal Behavioral State Dynamics in Depth-Related Locomotion in Mice

Understanding how mice process and respond to visual depth cues is crucial for studying visual perception, yet traditional behavioral analyses often miss key aspects of this process, such as the dynamic transitions between behavioral states, the influence of environmental context, and the integration of multiple spatial cues that shape depth-related behaviors. Here we demonstrate that mouse responses to visual depth cues are more sophisticated than previously recognized, involving both direct avoidance behaviors and complex modulations of exploratory patterns. By combining a modified circular apparatus with Hidden Markov Model analysis, we reveal that mice transition between three distinct behavioral states--resting, exploring, and navigating--in response to visual depth cues. Using this framework, we uncover several fundamental aspects of mouse visual processing: depth perception has an optimal range of spatial frequencies, with strongest responses to patterns between 6-8 cm; visual processing integrates multiple spatial cues rather than triggering simple avoidance; and initial strong cliff-avoidance responses evolve into more nuanced behavioral adaptations over time. Comparisons between wild-type C57BL/6J mice (Mus musculus), retinal degeneration models (rd1-2J, C57BL/6J background, Mus musculus), and control conditions confirm that these behavioral patterns specifically reflect visual processing rather than general exploratory behavior. These findings reveal that mouse depth perception involves sophisticated neural processing that modulates overall exploratory behavior rather than simply triggering avoidance responses. Our approach establishes a new framework for analyzing complex behavioral sequences in neuroscience research, demonstrating how refined behavioral analysis can reveal previously undetectable aspects of sensory processing.

animal behavior and cognition↗

bioRxiv2025

Genomic characterization of sub-populations in human pluripotent stem cell-derived retinal progenitor cells that drive retinal layer structure

The mechanism underlying retinal spheroid layer formation was investigated using Rax::GFP-positive retinal progenitor cells from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing revealed that well-layered spheroids transiently activated canonical WNT2B-FZD7 signaling followed by temporary expression of non-canonical WNT5A. Despite structural differences in vitro, non- layered and well-layered retinal spheroids on differentiation Day 60 successfully developed into cone and rod subtypes of photoreceptors and established synaptic connections with host bipolar cells after transplantation in a retinal degeneration rat model, resulting in light-evoked functional recovery. Additionally, part of the presumable Rax::GFP-positive retinal progenitor cells differentiated into non- retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium, and spinal cord-like tissues in vitro, reflecting the developmental plasticity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, whereas environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.

cell biology↗

bioRxiv2023

Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

Among the genome-editing methods for repairing disease-causing mutations resulting in dominant inhibition, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. For the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance of inherited retinal dystrophy, we improved the gene constructs available in both plasmid and adeno-associated virus (AAV) vectors, and established a workflow that uses in vivo electroporation to verify the in vivo efficacy. By targeting the mouse Rhodopsin gene, we derived a construct in which HITI-mediated gene insertion occurs in 80%-90% of transduced mouse rod photoreceptor cells. This construct suppressed degeneration and induced visual restoration in the mutant mice. The HITI-treatment constructs for the rhodopsin gene were shown to be effective in AAV vectors, and this construction is available for the mouse Peripherin 2 gene. These findings suggest that the workflow reported here may be useful for the generation of HITI-treatment constructs for various target genes and for the development of gene therapy products.

molecular biology↗

bioRxiv2022

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

neuroscience↗

bioRxiv2021

Tertiary lymphoid structures induced by CXCL13-producing CD4+ T cells increase tumor infiltrating CD8+ T cells and B cells in ovarian cancer

BackgroundTertiary lymphoid structures (TLSs) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. The presence of TLS is associated with a favorable prognosis in most solid malignancies. The recognition of the relevance of TLS to cancer has led to a growing interest in TLS as an immunomodulatory target to enhance tumor immunity, although how TLSs are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. MethodsTLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression were investigated in high grade serous ovarian cancer (HGSC) specimens. CXCL13 expression, which is strongly associated with TLS, and its localization in immune cells, were examined. We explored the tumor microenvironment for CXCL13 secretion by adding various inflammatory cytokines in vitro. The induction of TLS by CXCL13 was examined in a mouse model of ovarian cancer. ResultsCXCL13 gene expression correlated with TLS formation and the infiltration of T cells and B cells, and was a favorable prognostic factor for HGSC patients. The coexistence of CD8+ T cells and B-cell lineages in the TME was associated with a better prognosis of HGSC and was closely related to the presence of TLSs. CXCL13 expression was predominantly coincident with CD4+ T cells in TLSs and CD8+ T cells in TILs, and shifted from CD4+ T cells to CD21+ follicular dendritic cells as the TLS matured. Although TGF-{beta} was reported to stimulate CXCL13 production, our in vitro results revealed that CXCL13 secretion was promoted in CD4+ T cells under TGF-{beta} + IL-2-restricted conditions and in CD8+ T cells under TGF-{beta} + IL-12-rich conditions. In a mouse model of ovarian cancer, recombinant CXCL13 induced TLSs and enhanced survival by the infiltration of CD8+ T cells. ConclusionsTLS formation was promoted by CXCL13-producing CD4+ T cells and TLSs facilitated the coordinated antitumor responses of cellular and humoral immunity in ovarian cancer.

immunology↗

bioRxiv2020

Genetically engineered retina for improved retinal reconstruction after transplantation

ES/iPS-retinal sheet transplantation, which supplies photoreceptors as well as other retinal cells, has been shown able to restore visual function in mice with end-stage retinal degeneration. Here, by introducing a novel type of genetically engineered ES/iPS-retinal sheet with reduced numbers of secondary retinal neurons but intact photoreceptor cell layer structure, we reinforced the evidence that ES/iPS-retinal sheet transplantation can establish synaptic connections with the host, restore light responsiveness and reduce aberrant RGC spiking. Furthermore, we show that genetically engineered grafts can substantially improve the outcome of the treatment by improving neural integration. We speculate that this leads to reduced spontaneous activity in the host which in turn contributes to a better visual recovery.

neuroscience↗

bioRxiv2020

Capturing Human Trophoblast Development with Naive Pluripotent Stem Cells In Vitro

Trophoblast are extra-embryonic cells that are essential to maintain pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT) composing the placenta. Here we show that naive, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CT (nCT) recreated the human and monkey TE-to-CT transition. nCT self-renewed as CT stem cells and had the characteristics of proliferating villous CT and CT in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (pBAP), pBAP were distinct from nCT and human placenta-derived CT stem cells, exhibiting properties consistent of the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system will provide a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

developmental biology↗

bioRxiv2020

Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

Regenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

bioengineering↗

bioRxiv2020

Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals regulatory toggles operating in native and iPS-derived retina.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from native and iPSC-derived murine retina at four matched developmental stages spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal aggregates broadly recapitulate the native developmental trajectories. However, we show relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in iPSC-derived retina. We generate bulk ATAC-Seq data for native and iPSC-derived murine retina identifying [~]125,000 peaks. We combine single-cell RNA-Seq with ATAC-Seq information and obtain evidence that approximately half the transcription factors that are dynamically regulated during retinal development may act as repressors rather than activators. We propose that sets of activators and repressors with cell-type specific expression constitute "regulatory toggles" that lock cells in distinct transcriptome states underlying differentiation. We provide evidence supporting our hypothesis from the analysis of publicly available single-cell ATAC-Seq data for adult mouse retina. We identify subtle but noteworthy differences in the operation of such toggles between native and iPSC-derived retina particularly for the Etv1, Etv5, Hes1 and Zbtb7a group of transcription factors.

developmental biology↗