Biology subjects

Onishi, A.

Publications and source records attributed to Onishi, A..

8 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

Identification of a novel and divergent reptarenavirus in an Amazon coral snake (Micrurus spixii )

Reptarenaviruses are viruses belonging to the genus Reptarenavirus within the family Arenaviridae, which infect snakes and cause inclusion body disease (IBD), a fatal condition characterized by behavioral abnormalities and wasting. Although many reptarenaviruses have been identified thus far, the phylogenetic gaps between reptarenaviruses and the other arenaviruses suggest the existence of yet-to-be-identified reptarenaviruses filling the gaps. In this study, we identified a novel reptarenavirus from publicly available RNA-seq data derived from Amazon coral snake (Micrurus spixii) and tentatively named it Amazon coral snake virus 1 (ACSV-1). We identified four ACSV-1 contigs containing the putative full-length open reading frames of the NP, GP, and L genes, as well as the partial Z gene. Phylogenetic analyses showed that ACSV-1 is highly divergent from known reptarenaviruses. The NP, GP, and L genes showed 48.3%, 42.3%, and 45.7% nucleotide sequence identities, respectively, with those of the closest relatives. Based on the International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria, ACSV-1 can be assigned to a novel species of virus within the genus Reptarenavirus. This study expands our understanding of the diversity and evolution of reptarenaviruses.

microbiology↗

bioRxiv2025

Macromolecular Crowding-Affected Mobility of Bcnt/Cfdp1, a Disordered Component of the Srcap Complex, from the Nucleus to Cytosol in Subcellular Fractionation

Metazoan Bucentaur/Craniofacial Development Protein 1 (Bcnt/Cfdp1) is crucial for embryonic development, and its yeast homolog protein, SWC5, functions as part of the SWR1 complex in histone exchange reactions. Bcnt/Cfdp1, an intrinsically disordered protein (IDP) subject to various post-translational modifications, contains a putative nuclear localization sequence. Nevertheless, its subcellular localization remains ambiguous. We investigated its intracellular trafficking using diverse approaches. In C2C12 myoblasts, fluorescent protein-tagged Bcnt/Cfdp1 was predominantly localized within the nucleus, preferentially in regions of low DAPI density in myoblasts, and the localization persisted in differentiated multinucleated myotubes. Conversely, detergent-based biochemical fractionation of C2C12 and HEK293T cells indicated a substantial presence of Bcnt/Cfdp1 in cytoplasmic fractions. Quantitative proteomic analysis of fractionated proteins revealed that six IDPs, including a PEST-containing nuclear protein, demonstrated similar distribution patterns to Bcnt/Cfdp1; despite being annotated as nuclear proteins, they were detected in cytoplasmic fractions. Direct treatment of both cell lines with digitonin enabled the collection of released proteins in three steps to accurately isolate cytoplasmic proteins. Bcnt/Cfdp1 exhibited distinct extraction patterns compared to cytoplasmic soluble protein extracts in Ficoll/sucrose solution, while no differences were noted when cells underwent digitonin treatment in an isotonic solution. Quantitative proteomic analysis of these extracts highlighted the mobility or integrity of each protein within the cytoplasm. Consequently, although Bcnt/Cfdp1 is essential within the nucleus, our findings suggest its additional presence in the cytoplasm. We hypothesize that the elastic properties of Bcnt/Cfdp1 facilitate its translocation from the nucleus during subcellular fractionation, which is affected by macromolecular crowding.

biochemistry↗

bioRxiv2025

Evaluation of Candidate ''Kill or Cure'' Strategies to Treat MFN2-related Lipodystrophy

The R707W mutation in mitofusin 2, encoded by MFN2, causes a form of Multiple Symmetrical Lipomatosis (MFN2-MSL). This resembles sporadic, alcohol-associated MSL, combining loss of lower body adipose tissue with upper body adipose hyperplasia. Morbidity and sometimes mortality arise both from mechanical complications of head and neck adipose overgrowth, and metabolic complications. We reasoned that interventions that either mitigate the underlying cellular pathology, or that exacerbate it to induce selective death of hyperplastic adipose tissue may be beneficial. We thus assessed the effect of a metabolic or pharmacologic stressors or rapamycin in Mfn2R707W/R707W mice and or derived preadipocytes. 50mmol ethanol had little effect on WT or Mfn2R707W/R707W white preadipocytes, but increased mitochondrial content and blunted mitolysosome formation in Mfn2R707W/R707Wbrown preadipocytes. Daily consumption of 20% EtOH increased brown adipose tissue mass in female Mfn2R707W/R707W mice, and serum lactate in males. 200nM rapamycin - a candidate treatment - increased size and mitolysosome content of WT and Mfn2R707W/R707W white and brown preadipocytes, but these effects were blunted in Mfn2R707W/R707W cells. In male but not female Mfn2R707W/R707W mice, rapamycin reduced or reversed weight gain, reduced brown adipose mass, and increased serum Fgf21. Finally, a panel of other metabolic and pharmacological mitochondrial stressors solicited no selective death or ISR in Mfn2R707W/R707W preadipocytes. We conclude that ethanol mildly exacerbates MFN-MSL in mice, while rapamycin is tolerated. Lack of sensitisation to mitochondrial stressors implies that the MSL-inducing effect of MFN2 R707W may not be exerted through compromised oxidative phosphorylation.

physiology↗

bioRxiv2024

The metabolically protective energy expenditure increase of Pik3r1-related insulin resistance is not explained by Ucp1-mediated thermogenesis

Human SHORT syndrome is caused by dominant negative human PIK3R1 mutations that impair insulin-stimulated phosphoinositide 3-kinase (PI3K) activity. This produces severe insulin resistance (IR) and often reduced adiposity, commonly described as lipodystrophy. However unlike human primary lipodystrophies, SHORT syndrome does not feature fatty liver or dyslipidaemia. Pik3r1Y657*/WT (Pik3r1Y657*) mice metabolically phenocopy humans, moreover exhibiting increased energy expenditure. We have hypothesised that this increased energy expenditure explains protection from lipotoxicity, and suggested that understanding its mechanism may offer novel approaches to mitigating the metabolic syndrome. We thus set out to determine whether increased Ucp1-dependent thermogenesis explains the increased energy expenditure in Pik3r1-related IR. Male and female Pik3r1Y657* mice challenged with a 45% fat diet for 3 weeks at 21{degrees}C showed reduced metabolic efficiency not explained by changes in food intake or physical activity. No changes were seen in thermoregulation, assessed by thermal imaging and a modified Scholander protocol. Ucp1-dependent thermogenesis, assessed by norepinephrine-induced oxygen consumption, was also unaltered. Housing at 30{degrees}C did not alter the metabolic phenotype of male Pik3r1Y657* mice, but led to lowered physical activity in female Pik3r1Y657* mice compared to controls. Nevertheless these mice still exhibited increased energy expenditure. Ucp1-dependent thermogenic capacity at 30{degrees}C was similar in Pik3r1Y657* and WT mice. We conclude that the likely metabolically protective energy leak in Pik3r1-related IR is not caused by Ucp1-mediated BAT hyperactivation, nor impaired thermal insulation. Further metabolic studies are required to seek alternative explanations such as non Ucp1-mediated futile cycling. New and NoteworthyUnderstanding how Pik3r1Y657* mice and humans are protected from lipotoxicity despite insulin resistance may suggest new ways to mitigate metabolic syndrome. We find reduced metabolic efficiency and increased energy expenditure in Pik3r1Y657* mice but no differences in locomotion, thermoregulation or Ucp1-dependent thermogenesis. Protective energy expenditure in Pik3r1-related insulin resistance has an alternative, likely metabolic, explanation

physiology↗

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↗

bioRxiv2021

An intrinsic endothelial dysfunction causes cerebral small vessel disease

Small Vessel Disease (SVD) is the leading cause of vascular dementia, causes a quarter of strokes, and worsens stroke outcomes(1, 2). The disease is characterised by cerebral small vessel and white matter pathology, but the underlying mechanisms are poorly understood. Classically, the microvascular and tissue damage has been considered secondary to extrinsic factors, such as hypertension, consisting of microvessel stiffening, impaired vasoreactivity and blood-brain barrier dysfunction identified in human sporadic SVDs. However, increasing evidence points to an underlying vulnerability to SVD-related brain damage, not just extrinsic factors. Here, in a novel normotensive transgenic rat model where the phospholipase flippase Atp11b is deleted, we show pathological, imaging and behavioural changes typical of those in human sporadic SVD, but that occur without hypertension. These changes are due to an intrinsic endothelial cell dysfunction, identified in vessels of the brain white matter and the retina, with pathological evidence of vasoreactivity and blood-brain barrier deficits, which precipitate a secondary maturation block in oligodendroglia and myelin disruption around the small vessels. This highlights that an intrinsic endothelial dysfunction may underlie vulnerability to human sporadic SVD, providing alternative therapeutic targets to prevent a major cause of stroke and dementia.

neuroscience↗

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↗