Biology subjects

Chekuri, A.

Publications and source records attributed to Chekuri, A..

9 recordsLinked to original sources

bioRxiv2026

ELP1 Gene Augmentation Restores Visual Function in a Mouse Model of Familial Dysautonomia

Familial dysautonomia (FD) is an autosomal recessive sensory and autonomic neurodevelopmental and degenerative disorder characterized by complex neurological phenotypes. One of its most debilitating features is progressive optic neuropathy, which leads to severe visual impairment in FD patients by the third decade of life. Although several preclinical approaches have shown partial rescue of retinal ganglion cell (RGC) degeneration through increasing Elongator acetyltransferase complex subunit 1 (ELP1) expression in the retina, currently no treatments exist to prevent vision loss in FD. In this study, we performed a comprehensive analysis of visual function in a retina-specific FD mouse model (Pax6-Cre;Elp1loxp/loxp) and evaluated a gene supplementation strategy to restore human ELP1 protein levels in the retina. Longitudinal retinal assessments indicated that FD mice exhibit significant retinal nerve fiber layer (RNFL) thinning, as observed in FD patients. FD mice also showed reduced flash visual evoked potentials (VEPs), pattern electroretinography (pERGs), and photopic negative responses (phNRs) amplitudes, along with impaired visual acuity and contrast sensitivity, as assessed using optomotor response assay (OMR). Full-field electroretinography (ffERG) revealed reduced amplitude of dark-adapted a-waves, dark and light-adapted b-waves, indicating combined RGC and bipolar cell dysfunction. Intravitreal delivery of an adeno associated vector (AAV) vector (AAV2.U1a.hELP1) effectively restored physiological ELP1 protein expression, which resulted in a significant rescue of retinal structure and function. Gene supplementation with AAV2.U1a.hELP1 resulted in broad functional and structural improvement compared with untreated FD mice. In summary, our findings provide the first demonstration that ELP1 gene supplementation can effectively rescue RGC function in an FD mouse model and support AAV2.hELP1 at an optimized dose (5.4x108 vg) as a promising therapeutic approach for FD-associated optic neuropathy.

genetics↗

bioRxiv2025

AAV2-mediated intravitreal delivery of exon-specific U1 snRNA rescues optic neuropathy in a mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare autosomal recessive neurodegenerative disorder caused by a splicing mutation in the ELP1 gene. It predominantly affects the sensory and autonomic nervous systems, with progressive vision loss due to optic neuropathy being a universal and debilitating symptom. Retinal pathology in FD involves progressive thinning of the retinal nerve fiber layer (RNFL), resulting from the degeneration of retinal ganglion cells (RGCs). Notably, FD-associated vision loss has a postnatal onset, offering a critical window for therapeutic intervention before severe visual impairment develops in adolescence. Currently, no approved treatments exist to prevent or reverse vision loss in FD. In this study, we present a novel RNA-based therapeutic approach targeting ELP1 pre-mRNA splicing in the retina. We engineered exon-specific U1 small nuclear RNAs (ExSpeU1s) to enhance inclusion of exon 20 in the mutant ELP1 transcripts in the retina, thereby restoring full-length ELP1 expression. Delivery of ExSpeU1 via adeno-associated virus serotype 2 (AAV2) to the retina improved ELP1 splicing, rescued RGC loss, and visual function in an FD mouse model. These findings highlight ExSpeU1-mediated splicing correction as a promising therapeutic approach for treating optic neuropathy in FD, offering potential to preserve vision and improve quality of life for patients.

molecular biology↗

bioRxiv2025

Multi-omic analysis of photoreceptor alterations during early-onset retinal degeneration in Mfrp-/- mice

Loss-of-function mutations in the membrane frizzled-related protein (MFRP) gene are associated with autosomal recessive ocular disorders such as nanophthalmos, posterior microphthalmia, and retinitis pigmentosa, yet the molecular mechanisms underlying MFRP-related retinal degeneration remain poorly defined. To investigate these mechanisms, we generated and characterized Mfrp-/- mice and conducted an integrated multi-omic analysis to map the onset and progression of transcriptional changes during retinal degeneration. Using longitudinal in vivo imaging, histological and immunohistochemical analysis, qRT-PCR, single-nucleus RNA sequencing (snRNA-seq), snATAC seq (single-nucleus Assay for Transposase-Accessible Chromatin using sequencing) and spatial transcriptomics (GeoMx DSP), we identified early and progressive photoreceptor loss in Mfrp-/- mice, beginning by 28d. Retinal thinning and autofluorescent deposits in Mfrp-/- mice were accompanied by decreased expression of rod and cone opsins. Transcriptomic profiling by snRNA-seq revealed distinct, photoreceptor-specific gene expression changes, including early upregulation of inflammatory and stress-related genes and later downregulation of genes involved in chromatin remodeling, synaptic function, and neurodevelopment. Spatial transcriptomic analysis of the outer nuclear layer (ONL) revealed disruptions in mitochondrial function, mTORC1 signaling, and G protein-coupled receptor pathways. These findings define a temporally and spatially coordinated program of degeneration triggered by Mfrp loss and demonstrate that Mfrp is required for the maintenance of photoreceptor integrity and gene regulatory homeostasis during retinal development. Our work establishes Mfrp-/- mice as a robust model for dissecting photoreceptor vulnerability and provides a transcriptomic atlas of disease progression with potential relevance for therapeutic development in MFRP-associated retinal dystrophies.

genomics↗

bioRxiv2024

Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia

Familial dysautonomia (FD) is a fatal autosomal recessive sensory and autonomic neuropathy. FD is caused by a T-to-C point mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 (ELP1) gene, which results in tissue-specific skipping of exon 20 to cause a premature termination codon and thus redues ELP1 protein levels. Here, we developed a CRISPR-Cas-based cytosine base editing strategy to permanently correct the disease-causing mutation and restore canonical mRNA splicing. Through systematic engineering of base editors and guide RNAs, we identified an optimal editor configuration capable of achieving up to 70% on-target correction in human cells and that restored ELP1 exon 20 inclusion. To enable in vivo delivery, a dual adeno-associated virus (AAV) intein-split base editor was delivered via intravenous injections in a humanized FD mouse mode, resulting in genetic correction and significantly increased ELP1 exon 20 inclusion in the brain and other tissues. In FD patient-derived iPSC-sympathetic neurons, we observed [~]10% correction efficiency but rescued disease-associated neuronal hyperactivity, demonstrating that partial correction can restore functional phenotypes. Genome-wide analyses revealed minimal off-target editing across multiple human cell types, supporting the specificity of this approach. Together, these findings establish a precise and permanent genome editing strategy for FD and supports the development of a one-time disease-modifying therapy for FD and highlights the therapeutic potential of base editing for splicing disorders.

bioengineering↗

bioRxiv2023

A blood-brain-barrier penetrant AAV gene therapy rescues neurological deficits in mucolipidosis IV mice.

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits and progressive vision loss. Using AAV9 and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1-/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse that had exhibited significant degeneration at the time of the treatment. However, no increase of retinal thickness was observed after the gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.

genetics↗

bioRxiv2023

Transcriptome analysis in a humanized mouse model of familial dysautonomia reveals tissue-specific gene expression disruption in the peripheral nervous system.

Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1{Delta}20/flox. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.

genomics↗

bioRxiv2022

Development of a novel oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial Dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in the Elongator complex protein 1 gene (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1 protein, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 protein expression in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in patients. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound, PTC258, efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1{Delta}20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a two-fold increase in functional ELP1 protein in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

genetics↗

bioRxiv2021

Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage.

Elongator is a highly conserved protein complex required for transcriptional elongation, intracellular transport and translation. Elongator complex protein 1 (ELP1) is the scaffolding protein of Elongator and is essential for its assembly and stability. Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in ELP1 that lead to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD led to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for the formation and development of the nervous system. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression. Overall, this study highlights the crucial role of ELP1 during early embryonic neuronal development and reveals gene networks and biological pathways that are regulated by Elongator.

developmental biology↗

bioRxiv2020

A deep learning approach to identify new gene targets of a novel therapeutic for human splicing disorders

Pre-mRNA splicing is a key control point in human gene expression. Disturbances in splicing due to mutation or aberrant splicing regulatory networks lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of active and selective splicing modulator compounds have been recently identified, thus proving that pre-mRNA splicing is a viable target for therapy. We describe herein the identification of BPN-15477, a novel splicing modulator compound, that restores correct splicing of exon 20 in the Elongator complex protein 1 (ELP1) gene carrying the major IVS20+6T>C mutation responsible for familial dysautonomia. We then developed a machine learning approach to evaluate the therapeutic potential of BPN-15477 to correct splicing in other human genetic diseases. Using transcriptome sequencing from compound-treated fibroblast cells, we identified treatment responsive sequence signatures, the majority of which center at the 5 splice site of exons whose inclusion or exclusion is modulated by SMC treatment. We then leveraged this model to identify 155 human disease genes that harbor ClinVar mutations predicted to alter pre-mRNA splicing as potential targets for BPN-15477 treatment. Using in vitro splicing assays, we validated representative predictions by demonstrating successful correction of splicing defects caused by mutations in genes responsible for cystic fibrosis (CFTR), cholesterol ester storage disease (LIPA), Lynch syndrome (MLH1) and familial frontotemporal dementia (MAPT). Our study shows that deep learning techniques can identify a complex set of sequence signatures and predict response to pharmacological modulation, strongly supporting the use of in silico approaches to expand the therapeutic potential of drugs that modulate splicing.

bioinformatics↗