Targeting Mettl14 Using an RNA-Targeting Clustered Regularly Interspaced Short Palindromic Repeat-High-Fidelity Cas13x System Attenuates Doxorubicin-Induced Cardiotoxicity.

Background: Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, doxorubicin treatment is associated with cardiotoxicity, which limits its clinical use. Exercise can benefit both cancer and cardiovascular disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas13 (CRISPR-associated protein 13) platforms have emerged as effective technologies for targeting the expression of RNA in transcript levels. To develop exercise mimetics that can mimic the beneficial effects of exercise training to attenuate doxorubicin-induced cardiotoxicity, we are using the CRISPR-hf (high-fidelity)Cas13x system.

Methods: Adult male mice were swim-trained twice a day for 4 weeks to induce exercise-induced physiological cardiac hypertrophy. Adeno-associated virus 9-mediated METTL14 (methyltransferase-like 14) overexpression under the cardiac-specific ctnt promoter was used to overexpression METTL14 in vivo. RNA N⁶-methyladenosine inhibitor STM2457 was used to modulate global total RNA m⁶A levels in vivo. CRISPR-cr3-4/hfCas13x system was generated by hfCas13x guided crRNA3 and crRNA4 targeting the Mettl14 expressed under ctnt promoter and packaged in an adeno-associated virus 9.

Results: Swimming exercise alleviated doxorubicin-induced cardiotoxicity. METTL14 was increased in doxorubicin-treated hearts but decreased in exercised hearts. METTL14 overexpression inhibited exercise-induced physiological cardiac hypertrophy. Conversely, STM2457 treatment reversed the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Treatment with CRISPR-cr3-4/hfCas13x effectively inhibiting the expression of METTL14 in the heart, alleviating doxorubicin treatment-induced cardiac dysfunction and cardiac fibrosis.

Conclusions: Our results suggest that the CRISPR-hfCas13x system has the potential for generating exercise mimetics. Mimicking exercise by RNA-targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin-induced cardiotoxicity.