Biology subjects

Shamber, C. E.

Publications and source records attributed to Shamber, C. E..

2 recordsLinked to original sources

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↗

bioRxiv2024

AAV-mediated gene transfer of WDR45 corrects neurologic deficits in the mouse model of beta-propeller protein-associated neurodegeneration

Beta-propeller protein-associated neurodegeneration (BPAN) is an ultra-rare, X-linked dominant, neurodegenerative disease caused by loss-of-function mutations in the WDR45 gene. It manifests in neurodevelopmental delay and seizures followed by secondary neurologic decline with dystonia/parkinsonism and dementia in adolescence and early adulthood and is characterized by progressive accumulation of iron in the basal ganglia. WDR45 encodes {beta}-propeller-shaped scaffold protein, or WIPI4, which plays an important role in autophagosome formation. While the mechanisms of how WIPI4 loss of function results in neurologic decline and brain pathology have not yet been established, findings of lower autophagic activity provide a direct link between impaired autophagy and neurologic disease in BPAN. Here we performed phenotypical characterization of a novel mouse model of BPAN, WDR45_ex9+1g>a mouse. We identified hyperactive behavior and reduction of autophagy markers in brain tissue in WDR45_ex9+1g>a hemizygous males as early as at 2 months of age. Given the early onset and spectrum of neurologic symptoms such as hyper-arousal and attention deficits in human patients, this model presents a disease-relevant phenotype and can be used in preclinical studies. We used this mouse model for a proof-of-concept study to evaluate whether AAV-mediated CNS-targeted gene transfer of WDR45 can provide therapeutic benefit and be considered a therapeutic paradigm for BPAN. We observed successful expression of human WDR45 transcripts and WIPI4 protein in the brain tissue, rescue of hyperactive behavior, and correction of autophagy markers in the brain tissue. This data demonstrates that WDR45 gene transfer can be a promising therapeutic strategy for BPAN.

neuroscience↗