Biology subjects

Alves, C. R. R.

Publications and source records attributed to Alves, C. R. R..

5 recordsLinked to original sources

bioRxiv2026

Cardiac defects in spinal muscular atrophy and the role of SMN in cardiomyocyte homeostasis

Spinal muscular atrophy (SMA) is characterized by motor neuron degeneration caused by deficiency of the survival motor neuron (SMN) protein. However, evidence increasingly supports broader systemic involvement. This study aimed to examine cardiac pathology in SMA patients and to investigate how reduced SMN levels impact cardiomyocyte homeostasis. We analyzed postmortem data from 14 SMA type I patients from the pre-treatment era, integrating gross anatomical, histopathological, and clinical findings. To investigate cardiomyocyte-intrinsic effects of SMN deficiency, healthy human cardiomyocytes were subjected to SMN knockdown and assessed using metabolic assays and transcriptomic profiling. Key findings were further investigated in vivo using the Smn2B/- mouse model of SMA. We found heterogeneous cardiac involvement in SMA patients, including cardiomegaly, variable fat deposition and interstitial fibrosis. SMN knockdown in human cardiomyocytes induced a metabolic shift and widespread transcriptional dysregulation, with pathway analyses identifying selective upregulation of PTEN signaling. Elevated PTEN protein levels were observed in a subset of human SMA hearts and in early postnatal hearts of Smn2B/- mice. Our results demonstrate that the heart remains a biologically relevant target of SMN deficiency and highlights cardiomyocyte-specific metabolic and PTEN signaling alterations as potential contributors to cardiac involvement in SMA.

pathology↗

bioRxiv2025

Poison exon splicing in the human brain: a new frontier for understanding and targeting neurological disorders

BackgroundPoison exons (PE) are highly conserved exons whose inclusion creates premature termination codons (PTCs) and triggers nonsense-mediated decay (NMD) of the mature transcript. Despite their important role in post-transcriptional regulation, PEs remain poorly annotated due to the lack of systematic transcriptome-wide approaches. ResultsA comparative analysis of 957 eukaryotic transcriptomes revealed that Homo Sapiens exhibits the highest enrichment of NMD-targeted isoforms. By systematically predicting and annotating splicing events that generate PTCs, we found 9,814 PEs in the human genome. Using RNA-seq dataset from GTEx and BrainSpan, we analyzed PE splicing across tissues and developmental stages and identified 83 PEs uniquely found in the human brain, 973 PEs with brain-specific differential splicing, and 1,284 PEs differentially spliced during brain development. By integrating ClinVar variant annotations with SpliceAI predictions, we identified 1,625 pathogenic variants associated with neurological disorders that are predicted to affect the splicing of 743 PEs, and we functionally validated the impact of a subset of them using CRISPR prime-editing in human cells. Functional characterization of the deep intronic NDUFAF6 c.420+784C>T variant associated with Leigh syndrome demonstrated increased PE inclusion and a significant reduction in NDUFAF6 protein levels in iPSC-derived induced neurons. Mutant cells also exhibited impaired mitochondrial membrane potential compared to controls. Together, these findings provide functional evidence that the c.420+784C>T variant promotes aberrant PE inclusion, leading to reduced NDUFAF6 expression and mitochondrial dysfunction. ConclusionsOur findings highlight PEs as pivotal regulators of gene expression in the human brain and support the therapeutic targeting of PE splicing in neurological diseases.

genetics↗

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↗

bioRxiv2024

In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.

bioengineering↗

bioRxiv2023

Base editing as a genetic treatment for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C*G-to-T*A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A*T-to-G*C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

genetics↗