Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8)
Kaede Norota, Sen Ishizuka, Misa Hirose, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Saleh Ibrahim, Hideyoshi Harashima, Yuma Yamada
Abstract
Mitochondrial genome mutations are associated with various diseases and gene therapy targeted to mitochondria has the potential to effectively treat such diseases. Here, we targeted a point mutation in mitochondrial DNA (mtDNA) that can cause mitochondrial diseases via delivery of the clustered, regularly interspaced, short palindromic repeats/Cas9 (CRISPR/Cas9) system to mitochondria using an innovative lipid nanoparticle (LNP) delivery system. To overcome the major barrier of the mitochondrial membrane structure, we investigated a strategy to deliver ribonucleoprotein (RNP) directly to mitochondria via membrane fusion using MITO-Porter, a mitochondria-targeting lipid nanoparticle. First, we constructed RNP-MITO-Porter, in which an RNP was loaded into MITO-Porter using a microfluidic device. Sequence-specific double-strand breaks were confirmed when the constructed RNP-MITO-Porter was applied to isolated mitochondria. Next, the RNP-MITO-Porter was applied to HeLa cells, and a portion of the RNP-MITO-Porter was colocalized with mitochondria and caused sequence-specific double-strand breaks in mtDNA. Finally, RNP-MITO-Porter was successfully delivered to mitochondria of cells derived from a mouse carrying a point mutation (m.7778G > T) in mtDNA (mt-Atp8) (LMSF-N-MTFVB cells), and created double-strand breaks at the target sequence. RNP-MITO-Porter is expected to contribute significantly to the clinical application of mitochondrion-targeted gene therapy.