In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue

Abstract

Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.

Current Progress of Mitochondrial Genome Editing by CRISPR

“…The TAL effector proteins are linked to the I-TevI nuclease in mitoTev-TALE, rather than the FokI nuclease, as in mitoTALEN. Recently, a radically different approach for mtDNA editing has been reported, namely double-stranded DNA deaminase (DddA)-derived cytosine base editors (DdCBEs) ( Mok et al, 2020 ; Lee et al, 2021 ; Silva-Pinheiro et al, 2022 ). The DdCBE is composed of mitoTALE proteins, the interbacterial toxin DddA and an uracil glycosylase inhibitor.…”

Gene Therapy for Mitochondrial Diseases: Current Status and Future Perspective

“…Minczuk and collaborators successfully applied an in vivo proof-of-concept editing approach based on AAV-delivered DdCBE. They demonstrated the possibility of editing a specific mtDNA nucleotide in the heart of neonatal and adult mice [162], thus providing, for the first time, its potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes. However, the identification of low-frequency off-target editing in the nuclear genome of mouse embryos demonstrates the solid need to optimize DdCBE for specific base editing on mtDNA, especially before being used for treating mitochondrial diseases [163].…”

Prime time for base editing in the mitochondria

“…Mouse experiments by others also provide in vivo evidence of long-lasting mtDNA editing. 4 , 5 In summary, this exciting work is an important step towards extension of prime editing to mtDNA to allow all 12 possible base-to-base changes so that this technology can be used to develop more relevant disease models, increase our abilities to control cell functions and hopefully offer patients suffering with debilitating mitochondrial defects improved treatment options (Fig. 1 ).…”

“…A recent study showed the feasibility of in vivo mtDNA base editing in mouse hearts. 5 Using a cardio-tropic adeno-associated virus (AAV) serotype (AAV9.45) to deliver a DddA-derived cytosine base editor (DdCBE) to target the mouse mitochondrial Nd3 gene, the authors showed targeted editing of 10–20% in the cardiac tissue of mice analyzed 24-weeks after tail-vein injection of 1 × 10 12 AAV genomes per monomer per 8-week old mouse. 5 The mtDNA editing efficiency increased to 20–30% upon application of the same doses to neonatal mice via temporal vein injection.…”

“… 5 Using a cardio-tropic adeno-associated virus (AAV) serotype (AAV9.45) to deliver a DddA-derived cytosine base editor (DdCBE) to target the mouse mitochondrial Nd3 gene, the authors showed targeted editing of 10–20% in the cardiac tissue of mice analyzed 24-weeks after tail-vein injection of 1 × 10 12 AAV genomes per monomer per 8-week old mouse. 5 The mtDNA editing efficiency increased to 20–30% upon application of the same doses to neonatal mice via temporal vein injection. Thus, it will be interesting to explore editing efficiency upon in vivo delivery of mTALEDs, which are small enough to be encoded into the approximately 4.5 kb genetic cargo space of AAV vectors.…”