Mitochondrial adenine base editing of mouse somatic tissues via adeno-associated viral delivery
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eLife Assessment
The authors have demonstrated the use of adenine base editors delivered via adeno-associated viruses to introduce edits in the mitochondrial genome. The manuscript describes the methodology well, and the conclusions are convincingly supported by the results. The valuable results highlight the potential of these base editors to model mtDNA variations in somatic tissues in animal models.
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Abstract
The development of adenine base editing in mitochondria, alongside cytidine base editing, has significantly expanded the genome engineering capabilities of the mitochondrial DNA. We tested the recent advancements in adenine base editing technology using optimised TALEs targeting genes Mt-Cytb, Mt-CoII and Mt-Atp6 in mouse cells, and observed successful A:T to G:C conversions within the target windows of each gene. Then, we used the best performing pairs targeting the Mt-Atp6 gene to inject mice using adeno-associated viral delivery to post-mitotic tissue. We observed limited efficiency of adenine edits in mouse somatic tissue after 4 weeks, suggesting the necessity of further optimisation of this technology.
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eLife Assessment
The authors have demonstrated the use of adenine base editors delivered via adeno-associated viruses to introduce edits in the mitochondrial genome. The manuscript describes the methodology well, and the conclusions are convincingly supported by the results. The valuable results highlight the potential of these base editors to model mtDNA variations in somatic tissues in animal models.
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Reviewer #1 (Public review):
Summary:
This study represents an incremental step toward mitochondrial DNA editing but raises several concerns regarding its impact and broader applicability. The reported in vitro editing efficiency of 17% in mitotic cells, with non-specific editing across multiple A:T sites, offers limited improvement over prior technologies like DdCBE. Editing efficiency for the Mt-Atp6 gene was even lower (~4%), rendering it unlikely to produce functional changes relevant to mitochondrial function or bioenergetics.
While the modified TadA8e(V28R) mutant alleviated toxicity and enabled sufficient AAV production for in vivo experiments, the low in vivo editing efficiency (~4%) after 4 weeks was disappointing and unlikely to be biologically meaningful. Furthermore, the use of P1 postnatal tissues, which are still …
Reviewer #1 (Public review):
Summary:
This study represents an incremental step toward mitochondrial DNA editing but raises several concerns regarding its impact and broader applicability. The reported in vitro editing efficiency of 17% in mitotic cells, with non-specific editing across multiple A:T sites, offers limited improvement over prior technologies like DdCBE. Editing efficiency for the Mt-Atp6 gene was even lower (~4%), rendering it unlikely to produce functional changes relevant to mitochondrial function or bioenergetics.
While the modified TadA8e(V28R) mutant alleviated toxicity and enabled sufficient AAV production for in vivo experiments, the low in vivo editing efficiency (~4%) after 4 weeks was disappointing and unlikely to be biologically meaningful. Furthermore, the use of P1 postnatal tissues, which are still developing, raises questions about their suitability as models for postmitotic tissues, especially since the brain - a key organ affected by mitochondrial diseases - was excluded from the analysis.
Despite demonstrating feasibility for mitochondrial adenine base editing, the study highlights significant limitations, underscoring the need for further optimization. The reviewer also suggests adopting clearer terminology, such as "pathological variant" instead of "mutation," to enhance precision.
Strengths:
The study demonstrates the feasibility of adenine base editing in mitochondrial DNA, marking a step forward in expanding mitochondrial genome engineering capabilities. A notable strength is the development of a modified TadA8e(V28R) mutant, which successfully mitigated toxicity and enabled sufficient AAV production for in vivo experiments. This technical advancement addresses a key challenge in mitochondrial gene editing and provides a foundation for improving delivery methods and reducing off-target effects.
Additionally, the study highlights the potential for targeted mitochondrial DNA modifications using optimized TALEs, achieving A:T to G:C conversions in multiple genes. While the in vitro editing efficiency remains modest, the approach represents an important proof-of-concept for potentially advancing mitochondrial editing technologies, particularly in the context of addressing pathological variants.
Weaknesses:
The major weaknesses of the study center around its low editing efficiency, both in vitro and in vivo. In vitro editing achieved only 17% efficiency in mitotic cells, while the efficiency for the Mt-Atp6 gene was even lower, around 4%. This level of editing is unlikely to produce meaningful functional or biological changes, particularly in cells with pathological mtDNA variants. Similarly, in vivo, editing efficiency after a 4-week exposure period remained at approximately 4%, which is insufficient to support claims of effective mitochondrial genome editing. Another significant limitation is the lack of editing specificity, as observed changes occurred at multiple A:T sites within and across the editing window rather than being confined to a single position, raising concerns about precision and off-target effects.
The use of P1 postnatal mouse tissues also raises questions about the relevance of the model, as these tissues are still undergoing development and may not truly reflect postmitotic states. This casts doubt on whether the findings are transferable to mature tissues, such as the adult brain, which is frequently affected by mitochondrial diseases. Furthermore, the exclusion of brain tissue from the analysis limits the study's applicability to neurological disorders, a key area of mitochondrial disease research. The rationale for excluding brain tissue is not addressed, leaving an important gap in the study's scope.
The findings also lack novelty, as the reported low efficiency and lack of specificity are consistent with previous studies, making it unclear whether this work represents a significant advancement over existing technologies.
Collectively, these weaknesses underscore the need for further optimization of the approach, improved targeting specificity, and validation in more relevant models to demonstrate therapeutic potential.
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Reviewer #2 (Public review):
The authors have demonstrated the use of adenine base editors delivered via adeno-associated viruses to introduce edits in the mitochondrial genome. The manuscript describes the methodology well, and the conclusions are aptly supported by the results. It highlights the potential of these base editors to model mtDNA variations in somatic tissues in animal models.
However, there are a few comments that need to be addressed:
(1) Limitations of the small sample size need to be explained clearly for the results described.
(2) It will be beneficial for the readers if some light is shed on the possible reasons why the efficiencies of adenine base editing are lower than those reported for published cytosine base editors to introduce edits in the mitochondrial DNA.
(3) The conclusion should more explicitly address …
Reviewer #2 (Public review):
The authors have demonstrated the use of adenine base editors delivered via adeno-associated viruses to introduce edits in the mitochondrial genome. The manuscript describes the methodology well, and the conclusions are aptly supported by the results. It highlights the potential of these base editors to model mtDNA variations in somatic tissues in animal models.
However, there are a few comments that need to be addressed:
(1) Limitations of the small sample size need to be explained clearly for the results described.
(2) It will be beneficial for the readers if some light is shed on the possible reasons why the efficiencies of adenine base editing are lower than those reported for published cytosine base editors to introduce edits in the mitochondrial DNA.
(3) The conclusion should more explicitly address the limitations and future directions on low editing efficiency and what can be possible optimization steps.
(4) In Figure 1, A-to-G editing for the genes Mt-Cytb, Mt-CoII, and Mt-Atp6 appears to be strand-specific for the different architectures of adenine base editors. Do authors have a possible hypothesis if one of the strands is more favorable to editing depending on where the TadA8 binds or is it random?
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