Base editing of the GLB1 gene is therapeutic in GM1 gangliosidosis patient-derived cells.

GM1 gangliosidosis is an autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in the GLB1 gene, limiting the production of active lysosomal ß-galactosidase. Phenotypic heterogeneity is due in part to variant type, location within GLB1, and the amount of residual enzyme activity; in the most severe form, death occurs in infancy. With no FDA approved therapeutics, development of efficacious strategies for the disease is pivotal. CRISPR/Cas based approaches have revolutionized precision medicine and have been indispensable to the development of treatments for several monogenic disorders with bespoke strategies central to current research pipelines. We used CRISPR/Cas-adenine base editing to correct the GLB1 c.380G>A (p.Cys127Tyr) variant in patient-derived dermal fibroblasts compound heterozygous with the GLB1 c.481T>G (p.Trp161Gly) pathogenic variant. Nucleofection of plasmids encoding the target sgRNA and ABEmax restored the canonical guanine (32.2 ± 2.2 % of the target allele) and synthesis of active ß-galactosidase. Analysis of cellular markers of pathology revealed normalization of both primary glycoconjugate storage and lysosomal pathology. Furthermore, analysis of off-target sites nominated by the in silico tools Cas-OFFinder and/or CRISTA revealed no significant editing or indels. This study supports the use of CRISPR/Cas-based approaches for the treatment of GM1 gangliosidosis, and provides foundational data for future translational studies.

Generation of an infantile GM1 gangliosidosis induced pluripotent stem cell line (CHOCi005-A) for disease modeling and therapeutic testing.

GM1 gangliosidosis (GM1) is a rare autosomal recessive neurogenerative lysosomal storage disease characterized by deficiency of beta-galactosidase (ß-gal) and intralysosomal accumulation of GM1 ganglioside and other glycoconjugates. Resources for GM1 disease modelling are limited, and access to relevant cell lines from human patients is not possible. Generation of iPSC lines from GM1 patient-derived dermal fibroblasts allows for disease modelling and therapeutic testing in 2D and 3D cell culture models relevant to CNS disorders, including various neuronal subtypes and cerebral organoids. The iPSC line described here will be critical to therapeutic development and set the foundation for translational gene therapy work.

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.