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The development of curative treatments for mitochondrial diseases, which are often caused by mutations in mitochondrial DNA (mtDNA) that impair energy metabolism and other aspects of cellular homoeostasis, is hindered by an incomplete understanding of the underlying biology and a scarcity of cellular and animal models. Here we report the design and application of a library of double-stranded-DNA deaminase-derived cytosine base editors optimized for the precise ablation of every mtDNA protein-coding gene in the mouse mitochondrial genome. We used the library, which we named MitoKO, to produce near-homoplasmic knockout cells in vitro and to generate a mouse knockout with high heteroplasmy levels and no off-target edits. MitoKO should facilitate systematic and comprehensive investigations of mtDNA-related pathways and their impact on organismal homoeostasis, and aid the generation of clinically meaningful in vivo models of mtDNA dysfunction.

Mitochondria are complex factories that provide our cells with most of the energy we need to survive and perform daily tasks. They comprise their own small genome, mitochondrial DNA (mtDNA), which contains genes for parts of the energy-producing machinery. Mutations in mtDNA can lead to mitochondrial diseases, which are a devastating group of heterogenous inheritable diseases that can develop at any stage of life. Despite rapid developments in genome engineering for nuclear DNA, the incompatibility of certain techniques in mitochondria has meant that the field of mitochondrial genome modification has been impeded for many years. However, recent advances in mtDNA engineering techniques, such as programmable nucleases and base editors, will allow for a deeper understanding of the processes taking place in mitochondria and improve the prospects of developing treatments for mitochondrial diseases.

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.

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.

Mitochondria are subject to unique genetic control by both nuclear DNA and their own genome, mitochondrial DNA (mtDNA), of which each mitochondrion contains multiple copies. In humans, mutations in mtDNA can lead to devastating, heritable, multi-system diseases that display different tissue-specific presentation at any stage of life. Despite rapid advances in nuclear genome engineering, for years, mammalian mtDNA has remained resistant to genetic manipulation, hampering our ability to understand the mechanisms that underpin mitochondrial disease. Recent developments in the genetic modification of mammalian mtDNA raise the possibility of using genome editing technologies, such as programmable nucleases and base editors, for the treatment of hereditary mitochondrial disease.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies., Graphical Abstract Graphical AbstractThe A449T mutation in mouse POLγA, corresponding to the common A467T in patients, impairs the interaction between POLγA and B subunits, making POLγA amenable to degradation by LONP1 protease.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generated a POLGA449T/A449T mouse model, which reproduces the most common human recessive mutation of POLG, encoding the A467T change, and dissected the mechanisms underlying pathogenicity. We show that the A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ in vivo and in vitro. Interestingly, the A467T mutation also strongly impairs interactions with POLγB, the homodimeric accessory subunit of holo-POLγ. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA, which in turn exacerbates the molecular phenotypes of PolgA449T/A449T mice. Importantly, we validated this mechanism for other mutations affecting the interaction between the two POLγ subunits. We suggest that LONP1 dependent degradation of POLγA can be exploited as a target for the development of future therapies.

Leigh syndrome, or infantile necrotizing subacute encephalopathy (OMIM #256000), is one of the most common manifestations of mitochondrial dysfunction, due to mutations in more than 75 genes, with mutations in respiratory complex I subunits being the most common cause. In the present study, we used the recently described PHP.B serotype, characterized by efficient capacity to cross the blood-brain barrier, to express the hNDUFS4 gene in the Ndufs4−/− mouse model of Leigh disease. A single intravenous injection of PHP.B-hNDUFS4 in adult Ndufs4−/− mice led to a normalization of the body weight, marked amelioration of the rotarod performance, delayed onset of neurodegeneration, and prolongation of the lifespan up to 1 year of age. hNDUFS4 protein was expressed in virtually all brain regions, leading to a partial recovery of complex I activity. Our findings strongly support the feasibility and effectiveness of adeno-associated viral vector (AAV)-mediated gene therapy for mitochondrial disease, particularly with new serotypes showing increased permeability to the blood-brain barrier in order to achieve widespread expression in the central nervous system., Graphical Abstract, Systemically injected AAV-PHP.B efficiently crosses the blood-brain barrier in mice. In this study, a PHP.B-hNDUFS4 vector was used in both adult and newborn Ndufs4−/− mice, a model of mitochondrial neurodegeneration. Amelioration of the phenotype was observed in adults, but not newborns, due to low expression of the viral receptor in pups. Keywords:mitochondrial diseases, gene therapy, AAV, Ndufs4, complex I, OXPHOS, PHP.B, Leigh syndrome