Bs-452758-3 suppression-replacement gene therapy for -mediated cardiomyopathies

The LMNA gene encodes for nuclear lamina proteins Lamin A and C, which are critical for nuclear architecture maintenance, chromatin organization and gene expression. LMNA pathogenic mutations are associated with a range of pathologies in tissues, including the heart. Cardiac laminopathies manifest as structural changes and arrhythmias, which can lead to end-stage heart failure and sudden cardiac death. Current therapies for DCM-associated cardiomyopathies alleviate symptoms, without targeting the underlying molecular cause of the disease. To develop an LMNA suppression-replacement (SupRep) gene therapy that resolves the abnormal nuclear structure present in human cardiomyocytes (iPSC-CMs) harboring pathogenic LMNA mutations. Six shRNAs targeting LMNA were designed and evaluated for knockdown potency in HEK-TSA201 cells using RT-qPCR. SupRep LMNA gene therapy (lentivirus) was assembled by combining two expression cassettes into a single construct: 1) LMNA shRNA silences both endogenous alleles (Sup), and 2) an “shRNA-immune” (shIMM) LMNA cDNA (Rep). The LMNAH222P variant was inserted into an isogenic control (IC) iPSC-CM using CRISPR-Cas9. Nuclear structure of iPSC-CMs was analyzed on confocal images of cells mmune-stained with a-Lamin A/C antibody. Nuclei were assessed for three distinct defects: micronuclei, nuclear blebbing, and nuclear shrinking, both at baseline and after phenotype exacerbation with 6 hours of electrical stimulation (1 Hz, 5 msec, 10 V). The proportion of nuclear defects was quantified and analyzed using pair-wise Fisher’s exact tests with Holm-Bonferroni correction for multiple comparisons. The lead LMNA-targeting shRNAs suppressed LMNA expression by 75%. The proportion of anormal nuclei at baseline and following electrical stimulation was elevated in LMNAH222P compared to IC and was normalized following treatment (baseline: 19.73% in IC [n=1034] vs 24.87% in LMNAH222P [n=1319], [p=0.0068] vs 18.43% in LMNAH222P + SupRep [n=1134], [p=0.0003]; electrical stimulation: 25.46% in IC [n=974] vs 34.87% in LMNAH222P [n=1517], [p<0001] vs 25.14% in LMNAH222P + SupRep [n=1603], [p<0.0001]). We demonstrate the first proof-of-concept SupRep gene therapy for the correction of LMNA-mediated nuclear abnormalities in an iPSC-CM model of cardiac laminopathy. LMNA-SupRep gene therapy restored the impaired nuclear shrinking abnormality observed in a LMNA-H222P iPSC-CM.