Biology subjects

Lopes, M. M.

Publications and source records attributed to Lopes, M. M..

5 recordsLinked to original sources

bioRxiv2025

rAAV-Delivered Bicistronic Artificial microRNAs for Allele-Specific Silencing Improve Motor and Molecular Outcomes in Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats in the ATXN3 gene. This mutation induces a toxic gain-of-function of the ATXN3 protein, leading to neurodegeneration, particularly in the cerebellum and brainstem. Despite extensive research, no disease-modifying treatments are available for SCA3 patients. In this study, we developed and tested a novel therapeutic strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs designed to selectively silence the mutant ATXN3 allele. Through in vitro screening, we identified a lead construct (miATXN3-10x2) that effectively and specifically silenced the mutant allele by targeting of a single nucleotide polymorphism (SNP) associated with the repeat expansion. This construct was packaged into rAAV9 and delivered via intra-cerebellar administration into two mouse models of SCA3, resulting in robust suppression of mutant ATXN3 in the cerebellum. To assess long-term efficacy, we performed intra-cisterna magna (ICM) injections of rAAV9-miATXN3-10x2 in a severe SCA3 transgenic mouse model. Widespread distribution of viral vectors and miATXN3 copies was observed in disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor function at 5, 8, and 11 weeks post-injection. Histological analyses showed a reduction in mutant ATXN3 aggregates and a trend toward preventing shrinkage of cerebellar molecular layer. These findings were supported by dose-dependent reductions in mutant ATXN3 mRNA levels and decreased expression of neuroinflammatory markers in the cerebellum. Additionally, a significant increase of the neuronal marker NeuN was also observed in treated animals. Finally, transcriptomic profiling of the cerebellum demonstrated that treated transgenic animals exhibited an improved transcriptomic signature, shifting toward a wild-type profile. In conclusion, our findings highlight the therapeutic potential of a single administration of rAAVs encoding bicistronic artificial microRNAs for allele-specific gene silencing in SCA3. This study provides compelling preclinical evidence supporting the translation of this approach into clinical applications for SCA3 patients.

molecular biology↗

bioRxiv2025

AAV-mediated allele-specific silencing alleviates neuropathology in a novel non-human primate model of Spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of the cytosine-adenine-guanine (CAG) repeats in the ATXN3 gene. This mutation results in the production of an Ataxin-3 protein with an extended polyglutamine sequence, contributing to the diseases neuropathology. Currently, no treatment is available that can slow or halt the progression of SCA3. Gene-targeted therapies have gained significant attention for their potential to address the root cause of SCA3. Preliminary studies in transgenic mice using adeno-associated viral vector serotype 9 (AAV9) encoding artificial microRNAs targeting the mutant ATXN3 allele (AAV9-miR-ATXN3) have shown promising results. However, to advance this therapeutic approach toward clinical application, further studies in an animal model that more closely resembles human biology are essential. In this exploratory study, we assessed the biodistribution and target engagement of AAV9-miR-ATXN3 delivered via intracisterna magna (ICM) injection in non-human primates (NHPs). Using a lentiviral vector (LV) to introduce a mutant Ataxin-3 cDNA with 72 glutamines (LV-mutATXN3-Q72) into the NHP cerebellum, we successfully overexpressed SCA3 in the NHP brain. SCA3 NHP exhibited Ataxin-3 aggregation in the cerebellum, recruitment of inflammatory cells and reduced cerebellar volume. ICM administration of AAV9-miR-ATXN3 effectively directed transgene expression to key brain regions impacted by SCA3 pathology and enabled specific, dose-dependent silencing of mutant Ataxin-3. Furthermore, the therapeutic dose prevented the cerebellar morphological and biochemical alterations induced by the overexpression of mutant ATXN3. These proof-of-concept experiments are crucial, not only for advancing AAV9-miR-ATXN3 toward clinical use but also for establishing a valuable platform for validating future therapeutic interventions.

molecular biology↗

bioRxiv2025

The effect of environmental enrichment on immune cell DNA methylation profiles depends on the parity of sows.

The aim of this study was to identify epigenetic markers that reflected positive affective states in multiparous pregnant sows. The animals were housed during gestation in either a conventional (C) environment (2.4 m{superscript 2} per sow), featuring a concrete slatted floor and minimal enrichment, or in an enriched (E) environment (3.5 m{superscript 2} per sow) with deep straw bedding. Peripheral blood mononuclear cells (PBMCs) were isolated at day 98 of gestation (G98) and day 12 of lactation (L12) for genomic DNA extraction and reduced representation bisulfite sequencing (RRBS). Significant effects of individual class of parity (low-parity (LP) versus high-parity (HP)) were observed with the identification of 1,358 and 680 differentially methylated cytosines (DMCs) at G98 and L12, respectively. Interestingly, some of them are organized into differentially methylated regions (DMRs). Some DMRs colocalized with or near a gene and displayed a continuous methylation distribution (44 at G98 and 15 at L12 including 4 in common): 5 targeted genes are related to epigenetic regulation (DNMT3A, KDM8, HDAC4, SIRT2 and U2) and 12 to immune function (CD2, CD5, CAMK4, SECTM1, URODL1, CMIP, SEC14L1, SKI, TNFRSF1B, CCND3, SGK1, PACS1). These results suggested a true epigenetic impact of parity class on individual immunity. Considering the two parity classes separately, a minor effect of environmental enrichment was observed (at G98: 60 and 42 DMCs; at L12: 35 and 81 DMCs, in the LP and HP groups, respectively). Remarkably, some DMC-associated genes had previously been linked to affective states in humans. In conclusion, unexpected DNA methylation changes associated with parity class were identified, suggesting a genome adaptation during reproductive life and modifying the response to housing conditions. Furthermore, specific CpG sites emerged as potential biomarkers of positive affective states in pigs. HighlightsO_LIThis study revealed a substantial impact of the genetic background of sows on DNA methylation patterns, emphasizing the need to account for genetic factors when analysing epigenetic data. C_LIO_LIParity, which was confounded with the age of the sows in this study, significantly influenced DNA methylation, even among multiparous sows, highlighting the importance of this factor in modulating epigenetic mechanisms in immune cells. C_LIO_LIWe observed minor differences in the effects of environmental enrichment on DNA methylation profiles, which differed depending on the parity group. C_LIO_LIWe suggest a short list of possible biomarkers with biological meaning responding to environmental enrichment. C_LI

genomics↗

bioRxiv2025

Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALENs and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3.

neuroscience↗

bioRxiv2023

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

molecular biology↗