Biology subjects

Cheetham, M. E.

Publications and source records attributed to Cheetham, M. E..

14 recordsLinked to original sources

bioRxiv2026

Pharmacological rescue of cilia trafficking defects in IFT140 retinal organoid and RPE models of retinal dystrophy

Pathogenic variants in IFT140 are associated with a spectrum of syndromic and non-syndromic ciliopathies, with retinal degeneration as a common feature. Despite advances in understanding IFT140 function across various tissues, human retina-specific models are lacking. Here, we show that knock-in mice homozygous for the IFT140 patient variant c.932A>G (p.Y311C) did not develop retinal degeneration, while mice with the homozygous variant c.1451C>T (p.T484M), associated with non-syndromic retinal dystrophy, were embryonic lethal. Therefore, to understand the effect of these variants on retinal homeostasis, we generated novel human in vitro models of IFT140-associated retinal dystrophy, including CRISPR/Cas9 IFT140 knock-out (IFT140KO) induced pluripotent stem cells (iPSC) and patient-derived iPSC retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-ROs). IFT140KO iPSC-RPE cells display stubby cilia compared to isogenic controls, while IFT140T484M/T484Mpatient-derived iPSC-RPE cells exhibit slightly shorter cilia and cilia tip protein accumulation. Both IFT140KO and IFT140T484M/T484M iPSC-ROs show accumulation of cilia proteins at the connecting cilium and outer segment of photoreceptors, and mislocalization of rhodopsin to the inner segments and outer nuclear layer. Pharmacological screening of compounds previously reported to improve cilia structure identified the flavonoid eupatilin as the most effective molecule. Treatment with eupatilin improved cilium length and IFT traffic in iPSC-RPE, and IFT traffic and rhodopsin localization in iPSC-ROs. These findings emphasize the importance of human stem cell derived models to investigate tissue specific disease mechanisms and highlight the therapeutic potential of eupatilin to ameliorate cilia defects in retinal tissue.

neuroscience↗

bioRxiv2025

Antisense oligonucleotide allele-specific targeting of EFEMP1 in a patient-derived model of Doyne honeycomb retinal dystrophy

Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033C>T, p.Arg(345Trp) variant in EFEMP1, and is characterised by the early onset extracellular deposition of drusen between the retinal pigment epithelium basement membrane and underlying layers of Bruchs membrane. In this study, we developed an antisense oligonucleotide approach to target EFEMP1. We reprogrammed patient-derived renal epithelial cells to induced pluripotent stem cells followed by directed differentiation to retinal pigment epithelium and compared the phenotype to gene-corrected and EFEMP1 knockout patient-derived retinal pigment epithelium. In the patient-derived disease model, remodelling of the extracellular matrix occurred with progressive accumulation of extracellular deposits containing the drusen-associated proteins apolipoprotein E and collagen IV, in addition to EFEMP1. Moreover, the intracellular accumulation of neutral lipids was evident. We developed an allele-specific antisense oligonucleotide which specifically and effectively promoted the clearance of the EFEMP1 c.1033C>T transcript in the patient-derived disease model following assisted or gymnotic delivery. In this disease model, gymnotic delivery led to a decrease in extracellular deposits and cleared the intracellular accumulation of lipids, even after the onset of this disease phenotype, suggesting this could be a practical and effective therapeutic approach.

molecular biology↗

bioRxiv2025

Lost in communication: How Mueller glia cells fail to maintain retinal integrity in USH1C retinal organoids

Usher syndrome type 1, caused by pathogenic variants in the USH1C gene, leads to congenital deafness and progressive retinal degeneration resulting in vision loss. While auditory deficits can be compensated by cochlea implants and hearing aids, no treatment exists to prevent retinal degeneration. Here, we generated retinal organoids from induced pluripotent stem cells of two USH1C patients to elucidate the cellular and molecular mechanisms driving ocular pathogenesis. Single-cell RNA sequencing of both healthy and USH1C retinal organoids identified differential expression of genes related to phototransduction in photoreceptors, as well as alterations in cell adhesion and canonical Wnt signaling in Muller glia cells. Analysis of intercellular communication revealed an overall reduced signaling efficiency, particularly affecting Muller glia-mediated retinal adhesion processes. Morphological characterization of organoids confirmed transcriptome changes by showing degeneration of the outer limiting membrane and loss of adherens junction architecture. Moreover, photoreceptors revealed increased susceptibility to morphological and functional changes related to phototransduction. These results demonstrate that disruption of Muller glia signaling contributes to a loss of retinal integrity, providing novel insights into USH1C pathogenesis and offering targets for therapeutic interventions.

cell biology↗

bioRxiv2025

Preventing light-induced toxicity in a new mouse model of rhodopsin sector retinitis pigmentosa

Retinitis Pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive loss of rod photoreceptors. Sector RP is a form of RP, where degeneration originates in the inferior retina, mainly influenced by light exposure. Over 200 RHO variants are pathogenic and associated with autosomal dominant RP. RHOM39Ris one of the most common RHO variants linked to sector RP in the UK. A knock-in (KI) mouse model expressing RhoM39R was generated and characterized to investigate the mechanisms of degeneration associated with this variant and explore novel therapeutic strategies for rhodopsin sector RP. Under ambient light, RhoM39R/+ KI mice exhibited impaired retinal function by ERG, with some defects in OS ultrastructure, but retained normal outer nuclear layer (ONL) thickness. Repeated exposure to bright light led to photoreceptor loss. In contrast, RhoM39R/M39R KI mice in ambient light displayed severe retinal dysfunction, ONL thinning, and grossly abnormal OS ultra structure. In homozygous mice, a single bright light exposure significantly reduced ONL thickness within 48 h. The rescue of these models was achieved through reduced light exposure and pharmacological intervention. Rearing in dim red light (red cage condition) restored ERG responses in RhoM39R/+ KI mice and improved ONL thickness in RhoM39R/M39R KI mice. Transcriptomic analysis in RhoM39R/M39R KI mice revealed upregulation of Sphingosine 1-P Receptor (S1PR) transcripts. Treatment with the S1PR agonist Fingolimod (FTY720) before bright light exposure significantly reduced degeneration, demonstrating a protective effect in both heterozygous and homozygous models and suggesting potential a therapeutic approach for sector RP patients.

neuroscience↗

bioRxiv2024

Tissue-Specific Dynamics of TCF4 Triplet Repeat Instability Revealed by Optical Genome Mapping

Here, we demonstrate the utility of optical genome mapping (OGM) to interrogate the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) and gain novel insights into the tissue-specific nature of the disease. Genomic DNA (gDNA) samples derived from peripheral blood leukocytes and primary corneal endothelial cells (CECs) were analysed by OGM. Concurrently, all samples were genotyped by standard PCR-based methods to classify their expansion status. Individuals with one or more CTG18.1-expanded alleles ([≥]50 CTG repeats) detected in their leukocyte-derived gDNA were classified as expansion-positive. A customised bioinformatics pipeline was developed to perform CTG18.1-targeted OGM analysis. All linearised gDNA molecules containing labels flanking CTG18.1 were extracted, corrected for the repeats on the reference human genome and sized. Analysis of paired bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin, but displayed significantly higher levels of instability within the diseased corneal endothelium. Clusters of CTG18.1 molecules of approximately 1,800-11,900 repeats, beyond the ranges observed in individual-matched leukocyte samples, were detected in all CEC gDNA samples from expansion-positive cases. In conclusion, OGM is a powerful method to analyse the somatically unstable CTG18.1 locus. More generally, this work exemplifies the broader utility of OGM in exploring somatically unstable short tandem repeat loci. Furthermore, this study has highlighted the extreme levels of tissue-specific CTG18.1 somatic instability occurring within the diseased corneal endothelium, which we hypothesise plays a pivotal role in driving downstream pathogenic mechanisms of CTG18.1-mediated FECD.

genomics↗

bioRxiv2024

A proximity complementation assay to identify small molecules that enhance the traffic of ABCA4 misfolding variants.

ABCA4-related-retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2,200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30{degrees}C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.

molecular biology↗

bioRxiv2023

Eupatilin improves cilia defects in human CEP290 ciliopathy models

The photoreceptor outer segment is a highly specialized primary cilium essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290, there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290-related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids, by modulating the expression of rhodopsin, and by targeting cilia and synaptic plasticity pathways. This work sheds light into the mechanism of action of eupatilin, and supports its potential as a variant-independent approach for CEP290-associated ciliopathies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/536565v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@c2d45borg.highwire.dtl.DTLVardef@103c8b5org.highwire.dtl.DTLVardef@16e025eorg.highwire.dtl.DTLVardef@15d8be7_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Deciphering novel TCF4-driven molecular origins and mechanisms underlying a common triplet repeat expansion-mediated disease

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease. Authors summaryFuchs endothelial corneal dystrophy (FECD) leads to vision loss and is the most common repeat expansion-mediated disease characterised to date; most individuals with FECD harbour a non-coding CTG repeat expansion within the gene TCF4. FECD represents an important paradigm for other rare and devastating neurological repeat-mediated diseases, given its relatively mild and tissue-specific nature. Intriguingly, despite TCF4 being ubiquitously expressed, individuals with FECD only experience corneal disease, and the biological reason for this tissue-specificity remains elusive. Here, we use tissue from 31 individuals with FECD to perform complementary long-read, short-read and spatial transcriptomic analyses to enhance our understanding of mechanisms underpinning this disease. These data highlight that at least two mechanisms, RNA toxicity and TCF4 isoform dysregulation, underlie the disease state in affected corneal cells. Furthermore, TCF4 isoform skewing, with evidence of downregulation, suggests this mechanism in part may explain the unique vulnerability of the cornea. In addition, 7/134 FECD expansion negative cases were identified to harbour rare and potentially deleterious TCF4 variants, further supporting the hypothesis that dysregulation of TCF4 may be key to FECD pathophysiology. Biological insights presented here will guide the development of personalised FECD therapies and may inform the development of repeat-expansion mediated therapies more broadly.

molecular biology↗

bioRxiv2022

Antisense oligonucleotide therapy for the common Stargardt disease type 1-causing variant in ABCA4

The c.5461-10T>C p.[Thr1821Aspfs*6,Thr1821Valfs*13] variant has been identified as the most common severe Stargardt disease type 1 (STGD1)-associated variant in ABCA4. STGD1 is the most recurrent hereditary form of maculopathy and so far, no treatment is available for STGD1. In STGD1 patients homozygous for this variant, the onset of the disease typically is in childhood and patients are legally blind by early adulthood. The variant leads to exon skipping and generates out-of-frame ABCA4 transcripts that prevent the translation of functional ABCA4 protein. We applied antisense oligonucleotides (AONs) to restore the wild-type RNA splicing in ABCA4 c.5461-10T>C. The effect of AONs was investigated in vitro using an ABCA4 midigene model and 3D human retinal organoids (ROs) homozygous for the ABCA4 c.5461-10T>C variant. The mRNA in untreated ROs contained only disease-associated isoforms, whereas the organoids treated with the lead AON sequence showed 53% splicing correction and restoration of ABCA4 protein. Collectively, these data identified the lead candidate QR-1011 as a potent splice-correcting AON to be further developed as therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.

molecular biology↗

bioRxiv2021

Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

cell biology↗

bioRxiv2021

The role of Musashi-1 in CEP290 c.2991+1655A>G cryptic exon splicing in Leber Congenital Amaurosis

Human photoreceptors maximise alternative exon splicing to generate a unique set of gene isoforms. Conversely, the inclusion of a cryptic exon caused by the c.2991+1655A>G deep intronic change in CEP290 occurs in the human retina leading to Leber Congenital Amaurosis (LCA10). The RNA-binding protein Musashi-1 (MSI1) is a key component of alternative splicing in the developing mouse retina. Here we investigated the role of MSI1 in human photoreceptor-specific splicing and its potential role in CEP290 aberrant splicing disease. Alternative splicing was studied using human induced pluripotent stem cell derived 3D retinal organoid and RPE RNA-seq datasets and several photoreceptor gene isoforms were identified. Their temporal expression was resolved in control 3D retinal organoids in comparison to development and differentiation markers. Morpholino knockdown of MSI1 in control retinal organoids reduced the expression of several photoreceptor differentiation markers and the inclusion of photoreceptor-specific exons. Nonetheless, MSI1 knockdown in homozygous CEP290 c.2991+1655A>G LCA10 retinal organoids did not affect the inclusion of the LCA10-associated cryptic exon. These results show that while MSI1 is important for photoreceptor alternative splicing and homeostasis, it is not a major driver of the recognition of the CEP290 cryptic splice site and the manifestation of LCA10. HIGHLIGHTS{blacksquare} The human retina expresses a unique set of gene isoforms {blacksquare}Musashi-1 regulates alternative splicing in 3D human retinal organoids {blacksquare}Musashi-1 knockdown in 3D retinal organoids affects gene splicing and homeostasis in photoreceptors {blacksquare}Musashi-1 may regulate alternative splicing of cryptic exons in retina but not in LCA10

cell biology↗

bioRxiv2020

Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

Due to continuously high production rates of rhodopsin (RHO) and high metabolic activity, photoreceptor neurons are especially vulnerable to defects in proteostasis. A proline to histidine substitution at position 23 (P23H) leads to production of structurally misfolded RHO, causing the most common form of autosomal dominant Retinitis Pigmentosa (adRP) in North America. The AAA-ATPase valosin-containing protein (VCP) extracts misfolded proteins from the ER membrane for cytosolic degradation. Here, we provide the first evidence that inhibition of VCP activity rescues degenerating P23H rod cells and improves their functional properties in P23H transgenic rat and P23H knock-in mouse retinae, both in vitro and in vivo. This improvement correlates with the restoration of the physiological RHO localization to rod outer segments (OS) and properly-assembled OS disks. As a single intravitreal injection suffices to deliver a long-lasting benefit in vivo, we suggest VCP inhibition as a potential therapeutic strategy for adRP patients carrying mutations in the RHO gene.

neuroscience↗

bioRxiv2020

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2 associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. AAV mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.

neuroscience↗

bioRxiv2019

Symmetric arrangement of mitochondria:plasma membrane contacts between adjacent photoreceptor cells regulated by Opa1

Mitochondria are known to play an essential role in photoreceptor function and wellbeing that enables normal healthy vision. Within photoreceptors they are elongated and extend most of the length inner segment, where they supply energy for protein synthesis and the phototransduction machinery in the outer segment as well as acting as a calcium store. Here we examined the arrangement of the mitochondria within the inner segment in detail using 3D electron microscopy techniques and show they are tethered to the plasma membrane in a highly specialised arrangement. This includes mitochondria running alongside each other in neighbouring inner segments, with evidence of alignment of the cristae openings. As the pathway by which photoreceptors meet their high energy demands is not fully understood, we propose this to be a mechanism to share metabolites and assist in maintaining homeostasis across the photoreceptor cell layer. In the extracellular space between photoreceptors, Muller glial processes were identified. Due to the often close proximity to the inner segment mitochondria, they may too play a role in the inner segment mitochondrial arrangement as well as metabolite shuttling. OPA1 is an important factor in mitochondrial homeostasis, including cristae remodelling; therefore, we examined the photoreceptors of a heterozygous Opa1 knock-out mouse model. The cristae structure in the Opa1+/- photoreceptors was not greatly affected, but there were morphological abnormalities and a reduction in mitochondria in contact with the inner segment plasma membrane. This indicates the importance of key regulators in maintaining this specialised photoreceptor mitochondrial arrangement.

cell biology↗