Base editing rescues acid α-glucosidase function in infantile-onset Pompe disease patient-derived cells.

Infantile-onset Pompe disease (IOPD) results from pathogenic variants in the GAA gene, which encodes acid α-glucosidase. The correction of pathogenic variants through genome editing may be a valuable one-time therapy for PD and improve upon the current standard of care. We performed adenine base editing in human dermal fibroblasts harboring three transition nonsense variants, c.2227C>T (p.Q743∗; IOPD-1), c.2560C>T (p.R854∗; IOPD-2), and c.2608C>T (p.R870∗; IOPD-3). Up to 96% adenine deamination of target variants was observed, with minimal editing across >50 off-target sites. Post-base editing, expressed GAA protein was up to 0.66-fold normal (unaffected fibroblasts), an improvement over affected fibroblasts wherein GAA was undetectable. GAA enzyme activity was between 81.91 ± 13.51 and 129.98 ± 9.33 units/mg protein at 28 days post-transfection, which falls within the normal range (50-200 units/mg protein). LAMP2 protein was significantly decreased in the most robustly edited cell line, IOPD-3, indicating reduced lysosomal burden. Taken together, the findings reported herein demonstrate that base editing results in efficacious adenine deamination, restoration of GAA expression and activity, and reduction in lysosomal burden in the most robustly edited cells. Future work will assess base editing outcomes and the impact on Pompe pathology in two mouse models, Gaa c.2227C>T and Gaa c.2560C>T.

Base editing corrects the common Salla disease SLC17A5 c.115C>T variant.

Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the SLC17A5 gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, SLC17A5 c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous SLC17A5 c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the Slc17a5 c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant SLC17A5 c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.

GM1 Gangliosidosis: Mechanisms and Management.

The lysosomal storage disorder, GM1 gangliosidosis (GM1), is a neurodegenerative condition resulting from deficiency of the enzyme ß-galactosidase (ß-gal). Mutation of the GLB1 gene, which codes for ß-gal, prevents cleavage of the terminal ß-1,4-linked galactose residue from GM1 ganglioside. Subsequent accumulation of GM1 ganglioside and other substrates in the lysosome impairs cell physiology and precipitates dysfunction of the nervous system. Beyond palliative and supportive care, no FDA-approved treatments exist for GM1 patients. Researchers are critically evaluating the efficacy of substrate reduction therapy, pharmacological chaperones, enzyme replacement therapy, stem cell transplantation, and gene therapy for GM1. A Phase I/II clinical trial for GM1 children is ongoing to evaluate the safety and efficacy of adeno-associated virus-mediated GLB1 delivery by intravenous injection, providing patients and families with hope for the future.

Electrostatic Complementarity Drives Amyloid/Nucleic Acid Co-Assembly.

Proteinaceous plaques associated with neurodegenerative diseases contain many biopolymers including the polyanions glycosaminoglycans and nucleic acids. Polyanion-induced amyloid fibrillation has been implicated in disease etiology, but structural models for amyloid/nucleic acid co-assemblies remain limited. Here we constrain nucleic acid/peptide interactions with model peptides that exploit electrostatic complementarity and define a novel amyloid/nucleic acid co-assembly. The structure provides a model for nucleic acid/amyloid co-assembly as well as insight into the energetic determinants involved in templating amyloid assembly.