These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology, underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.
Abstract
Background LMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. Results In this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. Conclusions Our findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.
Hereditary spherocytosis (HS) is the most common inherited chronic hemolytic anemia and results from defects in proteins of the erythrocyte membrane skeleton. Nonsense mutations in ANK1 are a common cause of HS, yet current treatments remain largely supportive without addressing genetic defects. In this study, we identified two ANK1 nonsense mutations (p.R281X and p.Q744X) in unrelated HS pedigrees and generated K562 erythroid differentiation models using cytosine base editor-mediated knockin to evaluate potential targeted therapies. Functional analyses confirmed that both mutations markedly reduced ankyrin-1 expression and disrupted membrane-skeletal integrity. We then assessed two targeted therapeutic strategies: translational readthrough-inducing drugs (TRIDs) and adenine base editors (ABEs). Gentamicin promoted translational readthrough at both mutant sites, whereas CC-90009 showed activity primarily at p.Q744X. Both agents partially restored full-length ankyrin-1 expression, accompanied by restored membrane stability. In parallel, ABE8e-mediated correction achieved efficient (>85%) and precise genomic repair in bulk-edited K562 populations, leading to near-complete recovery of ankyrin-1 expression and membrane-skeletal integrity with minimal off-target effects. Together, these findings provide exploratory proof-of-concept for targeted molecular intervention in ANK1 nonsense mutation-associated HS, highlighting TRIDs and ABEs as mechanistically distinct strategies with complementary strengths and limitations and suggesting their relative utility may depend on mutation-specific and clinical contexts.
Shan Li, Juan Li, Wenjing Shi et al.· Molecular Therapy: Nucleic A...· 0 citations
Background LMNA codes a widely expressed nuclear cytoskeletal protein (lamin A/C) with multiple important functions. Pathogenic LMNA genetic variation may lead to autosomal dominant cardiomyopathy, though the severity and rate of progression can vary with the specific nucleotide change and location. Prior studies showed that induced pluripotent stem cells (iPSC)-derived cardiomyocytes (iCMs) with LMNA R541C exhibited reduced LMNA protein abundance, increased sarcomere disorganization, and abnormal electrophysiology. Methods We investigated the LMNA-R541C variant that exhibits a highly penetrant and severe clinical cardiomyopathy phenotype using transcriptomic analysis of iCMs. Patient-derived iPSCs with CRISPR-corrected (clustered regularly interspersed short palindromic repeats) isogenic control cells and CRISPR knock-in LMNA-R541C heterozygous iPSCs were generated for isogenic controlled experiments. Results In differential gene expression analyses we observed that LMNAR541C/WT iPSC-derived cardiomyocytes had consistent perturbations in 123 genes across CRISPR-corrected and knock-in experiments compared to controls. Pathway analysis identified that the G2M checkpoint and oxidative phosphorylation processes were consistently dysregulated and confirm these findings in previously published iPSC and murine models. Discussion These results implicate perturbed gene expression and pathways that may contribute to the severe phenotypes in LMNA-R541C. Informatic analysis of pathways suggests several drug classes including multiple cardiac glycosides as potential targeted therapeutic candidates to be explored.
Thomas E. Keller, Ci Koehring, Brett W. Higgins et al.· bioRxiv· 0 citations
Variants in
LMNA
are established causes of inherited dilated cardiomyopathy (DCM); however, the clinical significance of many rare missense variants remains uncertain. We identified the rare
LMNA
c.929 A > G (p.Gln310Arg) variant in a patient with progressive DCM and performed preliminary variant-specific functional characterization in an AC16 transient overexpression model.
Duo whole-exome sequencing and copy-number variation analysis were performed in the proband and his son. Wild-type and p.Gln310Arg lamin A expression constructs were transiently transfected into AC16 cells. Flag-tagged protein expression, Flag-based transfection efficiency, and lamin A/C distribution were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential and intracellular ROS-associated fluorescence were assessed using JC-1 and DCFH-DA staining, respectively. ERK1/2, JNK, and p38 MAPK phosphorylation was evaluated by western blotting. Hypertrophy-associated
NPPA
and
NPPB
expression was measured by RT-qPCR, and cell area was quantified after phalloidin staining.
The
LMNA
c.929 A > G variant was classified as a variant of uncertain significance. Wild-type and p.Gln310Arg lamin A constructs showed comparable Flag-tagged protein expression and Flag-based transfection efficiency. Expression of p.Gln310Arg lamin A was associated with an increased proportion of cells displaying ring-like lamin A/C staining patterns. Variant-expressing cells showed increased intracellular DCF fluorescence, reduced JC-1 red-to-green fluorescence ratio, and selectively increased ERK1/2 phosphorylation, whereas no significant changes were detected in JNK or p38 MAPK phosphorylation. The same cells also showed a hypertrophy-like phenotype characterized by increased
NPPA
and
NPPB
expression and enlarged cell area.
In an AC16 transient overexpression model, expression of p.Gln310Arg lamin A was associated with altered lamin A/C distribution, increased intracellular ROS-associated fluorescence, reduced mitochondrial membrane potential, selectively increased ERK1/2 phosphorylation, and a hypertrophy-like cellular phenotype. These findings provide preliminary variant-specific functional evidence but do not establish the pathogenicity of the variant or the causal relationships among the observed cellular abnormalities. Further validation in endogenous and physiologically relevant cardac models is required.
Zikang Han, Yonghao Sheng, Hanice Sun et al.· Hereditas· 0 citations
The first functional characterization of the cardiomyopathy-associated SMYD1 N101S variant identified in a child with severe infantile cardiomyopathy is provided, establishing a mechanistic link between SMYD1 dysfunction and infantile cardiomyopathy and highlighting the importance of integrating genomic and functional approaches in rare cardiovascular disease.
Marta W. Szulik, Clint Gwynn, Magnus Creed et al.· bioRxiv· 0 citations
It is demonstrated that expanded DMPK transcript levels modulate free MBNL1 concentration and alternative splicing in a dose-dependent manner, underscoring the central role of repeat RNA expression in DM1 pathogenesis.
Lise Ripken, Thomas D. Hoekman, M. Willemse et al.· Human Molecular Genetics· 0 citations
These findings support the value of preclinical models for translational studies, and autophagy enhancement as a potential therapeutic strategy for HSPB8-related myopathy, and evaluated trehalose, a natural disaccharide that induces HSPB8 and enhances autophagy.
Alyaa Shmara, Lan Weiss, A. Gromova et al.· bioRxiv· 0 citations
