RESUMO
BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.
RESUMO
Non-ferrous metal smelting emits large amounts of organic compounds into the atmosphere. Herein, 20 parent polycyclic aromatic hydrocarbons (PPAHs), 9 nitrated PAHs (NPAHs), 14 chlorinated PAHs (ClPAHs), and 6 alkylated PAHs (APAHs) in atmospheric samples from a typical non-ferrous metal smelting plant (NMSP) and residential areas were detected. In NMSP, benzo[a]pyrene, dibenz[a,h]anthracene, 6-nitrochrysene, 9-chlorofluorene, and 1-methylfluorene were the predominant compounds in the particulate phase, while phenanthrene constituted 57.3% in the gaseous phase. The concentration of PAHs in residential areas around NMSP was 1.8 times higher than that in the control area. Additionally, there was a significant negative correlation between the concentration and the distance from the NMSP. In terms of health risks, although the skin penetration coefficient of PM2.5 is smaller than that of the gaseous phase, dermal absorption of PM2.5 posed a greater threat to the population, the incremental lifetime cancer risk (ILCR) of NMSP was 1.8 × 10-4. After considering bioavailability, BILCR decreased by 1-2 orders of magnitude in different regions, and dermal absorption decreased more than inhalation intake. Nevertheless, the dermal absorption of PM2.5 in NMSP still presents a probable carcinogenic risk. This study provides a necessary reference for the subsequent control of NMSP contamination.