Gene Therapy for Heart Failure: Impact on Mitochondrial Dysfunction

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have significantly improved their manuscript. From my point of view, the manuscript is acceptabel for publication.

Author Response

Response to Reviewer 1

Comments and Suggestions for Authors

Comment: The authors have significantly improved their manuscript. From my point of view, the manuscript is acceptable for publication.

Response: We sincerely thank the Reviewer for the positive evaluation of our manuscript and for acknowledging the substantial improvements made in the revised version. We greatly appreciate the Reviewer’s assessment that the manuscript is suitable for publication.

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Blagonravov and colleagues' review focuses on gene therapy for heart failure using AAV vectors to correct mitochondrial dysfunction. The authors demonstrate a deep understanding of the molecular mechanisms of pathogenesis, including metabolic switching, mitochondrial dynamics, and the role of NAD+/sirtuin signaling. The review is well organized. The authors logically move from the pathophysiological foundations (Section 2) to molecular targets (Section 3), characteristics of AAV vectors (Section 4), and development prospects (Section 5). The review provides high-quality illustrations, and the authors draw on a sufficiently broad range of relevant literature for 2023-2025. The authors appropriately discuss the limitations of current approaches, including the critical importance of administration timing, differences between rodents and large animals, and immunogenicity issues. The review's practical focus is also noteworthy, evident in its thorough description of delivery methods (systemic, intracoronary, MCARD) and inclusion of clinical data from CUPID/CUPID 2. Despite the overall positive impression, I have a number of critical comments:

1. The authors insufficiently discuss clinical translation issues. Information on CUPID 2 is scant, and there is no analysis of why other targets (CPT1B, CAV3) have not reached the clinic.
2. The immune response is only briefly addressed. The authors mention neutralizing antibodies to AAV9, but there is virtually no discussion of the CD8+ T-cell response against transgenic cardiomyocytes or immune tolerance strategies.
3. The review lacks an analysis of sex differences. Disease staging (which targets are effective at which stages) is also only superficially discussed.
4. I recommend that the authors add a table systematically comparing the transduction of different AAV serotypes depending on the animal model, administration method, and dose.

Author Response

Response to Reviewer 2

Comments and Suggestions for Authors

Comment: Blagonravov and colleagues' review focuses on gene therapy for heart failure using AAV vectors to correct mitochondrial dysfunction. The authors demonstrate a deep understanding of the molecular mechanisms of pathogenesis, including metabolic switching, mitochondrial dynamics, and the role of NAD+/sirtuin signaling. The review is well organized. The authors logically move from the pathophysiological foundations (Section 2) to molecular targets (Section 3), characteristics of AAV vectors (Section 4), and development prospects (Section 5). The review provides high-quality illustrations, and the authors draw on a sufficiently broad range of relevant literature for 2023-2025. The authors appropriately discuss the limitations of current approaches, including the critical importance of administration timing, differences between rodents and large animals, and immunogenicity issues. The review's practical focus is also noteworthy, evident in its thorough description of delivery methods (systemic, intracoronary, MCARD) and inclusion of clinical data from CUPID/CUPID 2. Despite the overall positive impression, I have a number of critical comments:

Response: We sincerely thank the Reviewer for the positive and detailed evaluation of our manuscript and for the constructive comments. We have carefully addressed all points raised and substantially revised the manuscript accordingly. Below we provide a point-by-point response. All changes and additions, including newly added references, have been highlighted in color in the revised manuscript.

 

Specific Comments:

Comment 1: The authors insufficiently discuss clinical translation issues. Information on CUPID 2 is scant, and there is no analysis of why other targets (CPT1B, CAV3) have not reached the clinic.

Response 1: We agree with the Reviewer that a more comprehensive discussion of clinical translation challenges is essential for a review focused on gene therapy for heart failure. In response, we have expanded Section 5 (“Conclusions and Perspectives”) by adding a two-paragraph discussion addressing the outcomes of the CUPID 2 trial and the multifactorial reasons why several promising preclinical targets, including CPT1B and CAV3, have not yet progressed to clinical trials (lines 649–682).

Comment 2: The immune response is only briefly addressed. The authors mention neutralizing antibodies to AAV9, but there is virtually no discussion of the CD8+ T-cell response against transgenic cardiomyocytes or immune tolerance strategies.

Response 2: We are grateful to the Reviewer for raising this important point. In the revised manuscript, we have expanded Section 4 (“Modern Characteristics of AAV Vectors for Cardiomyocytes”) to include a detailed discussion of innate immune sensing of AAV vectors (including NF-kB and TLR9 signaling), the formation of neutralizing antibodies, MHC-mediated activation of capsid- and transgene-specific CD8+ T cells as well as current immune tolerance strategies (lines 487–519).

Comment 3: The review lacks an analysis of sex differences. Disease staging (which targets are effective at which stages) is also only superficially discussed.

Response 3: We thank the Reviewer for this valuable suggestion. In response, we have expanded Section 3 (“Mitochondrial Targets for Viral and Nonviral Vectors in CMCs to Reduce HF”) to provide a more detailed analysis of both stage-dependent targeting of mitochondrial abnormalities in HF and sex-specific differences in mitochondrial dysfunction, with implications for gene therapy (lines 295–343).

Comment 4: I recommend that the authors add a table systematically comparing the transduction of different AAV serotypes depending on the animal model, administration method, and dose.

Response 4: We sincerely appreciate the Reviewer’s helpful recommendation. Accordingly, we have added a new table to Section 4 (“Modern Characteristics of AAV Vectors for Cardiomyocytes”) entitled “Comparative cardiac transduction efficiency of various AAV serotypes across animal models, delivery methods and dosing regimens” (Table 2). This table systematically summarizes the differences in cardiac transduction efficiency among AAV serotypes, animal models, routes of administration, and vector doses (line 623–624).

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors responded adequately to my comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript has been thoroughly evaluated and requires minor revisions before it can be considered for publication. Substantial modifications are necessary to improve its quality and relevance for acceptance. Moreover, the manuscript contains multiple grammatical, spelling, and language issues that should be corrected to ensure clarity and accuracy.

1) Figures 1 and 2 are referenced but not fully explained; include brief captions or sentences describing their experimental significance and what they demonstrate.

2) Ensure that all terms and abbreviations are used consistently throughout the manuscript. For instance, ‘heart failure’ should always be abbreviated as HF, and ‘cardiomyocytes’ as CMCs. Define each abbreviation at first use and maintain the same form thereafter.

3) The manuscript adequately summarizes molecular pathways (e.g., PGC-1α, PPARα), but some explanations could be streamlined to maintain focus on gene therapy rather than general mitochondrial physiology.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Gene Therapy for Heart Failure: Impact on Mitochondrial Dysfunction” by Mikhail Blagonravov et al., according to Author’s claim “summarizes pathophysiological mechanisms associated with mitochondrial dysfunction, which is mainly caused by increased oxidative stress and impaired mitochondrial biodynamics under HF progression. It also addresses possible ways to modulate these processes using gene therapy. Special attention is paid to modern characteristics of AAVs that can be used as vectors for the efficient delivery of desired genes to CMCs.”

In my opinion, this review in its current state requires significant revision. The various chapters are uneven in quality and the richness of useful information. The review contains numerous controversial and ambiguous statements. When reading some sections, one gets the feeling that the authors didn't look beyond the abstracts of the cited articles.

Specific Comments

1. The first and one of the main shortcomings of the review is the insufficient immersion into the problem of mitochondrial dysfunction, which arises as a consequence or as a cause of heart failure. In my opinion, before discussing solutions to this problem, its causes need to be better understood. I would recommend that the authors better understand and better structure the mechanisms underlying mitochondrial dysfunction. Perhaps it would be best to begin with a classification of heart failure mechanisms.
2. The Authors' narrative is replete with unnecessary repetitions and a lack of relevant information:

        Abstract. Mitochondrial dysfunction, a distinctive feature of HF, leads to a progressive decrease in bioenergetic reserves due to switching of energy production from oxidation of fatty acids in mitochondria to glycolytic pathways.

• Lines 49-51 “Mitochondrial dysfunction, a hallmark of HF, results in a progressive reduction in bioenergetic reserves due to the shift in energy production from mitochondrial fatty acid oxidation to glycolytic pathways. This process leads to a decrease in fatty acid oxidation [4]”
• Lines 90-94 “Mitochondrial dysfunction is a common feature of HF and can be either a consequence or cause of the condition [4]. HF exhibits a maladaptive response, a progressive reduction in bioenergetic resources, a shift in energy generation from fatty acid oxidation within mitochondria to glycolytic routes…”
• Lines 97-99 “Mitochondrial dysfunction, which occurs in patients with HF, is associated with an unadaptive response and a progressive decline in bioenergetic reserves, irrespective of the etiology of the condition [2].”
• Lines 151-153 “The pathological processes that occur in heart failure result in mitochondrial dysfunction. This is due to a disruption in intracellular energy metabolism caused by a change in the oxidation substrate, leading to substrate hypoxia.”
• Lines 224-226 “Another important pathogenetic mechanism of HF is mitochondrial dysfunction [2]. Mitochondrial dysfunction leads to dysregulation of calcium levels, oxidative stress, protein toxicity, and cardiomyocyte death [8]”.

3. The authors discuss the mitochondrial quality control system, the reorganization of energy metabolism, mtDNA damage and loss caused by oxidative stress, and the deficiency of key proteins. What comes first: mtDNA mutations, ROS generation, or disruption of cytosolic Ca2+ homeostasis? What leads to the reorganization of bioenergetics? The activity of which proteins is critical for the development of heart failure and mitochondrial dysfunction, and which are a consequence of them? In its current form, this review does not answer these questions. Therefore, potential targets for genetic manipulation are completely unclear.
4. Mitochondrial Biogenesis in Heart Failure. Lines 105-122. The authors list a series of facts that don't add up to a complete picture for the less-versed reader. Why is fatty acid oxidation impaired? Why doesn't switching to glucose metabolism, which is readily oxidized by mitochondria as pyruvate, save cellular bioenergetics while ketone bodies do? These important points require clarification. What is the significance of "Increased acetylation of mitochondrial proteins (BCAAs oxidation), including pyruvate and succinate dehydrogenases, malate-aspartate shuttle pathway, the tricarboxylic acid cycle, and enzymes involved in fatty acid oxidation, has been observed in HF models"? Could this somehow be related to the inability to oxidize glucose via oxidative phosphorylation? Could sirtuins exert a protective effect in this context? How is acetylation related to mPTP opening? It is well known that matrix NADH is a potent inhibitor of its opening. How does the mitochondrial quality control machinery "aid in mitochondrial DNA repair" (Line 148)? Figure 1 is completely uninformative. Does ATP cause an increase in ROS or vice versa? The link to the diagram appears in the context of fission/fusion, but neither is present in the diagram. What do the authors mean in Lines 177-197? It is difficult to deduce from their reasoning, and the conclusion "Therefore, by modulating the optimal balance of mitochondrial activity, it is possible to improve mitochondrial function, delay the onset of left ventricular dysfunction, and slow the progression of HF" is hardly meaningful.
5. Mitochondrial Targets for Viral and Nonviral Vectors in CMCs to Reduce Heart Failure. Which mitochondrial proteins are deficient in heart failure? Can Serca2a, S100A1, Beta3 adrenergic receptor, and, strictly speaking, caveolin 3 be considered "mitochondrial targets"? Can any quantitative metrics be provided for the efficacy of various AAVs?
6. Modern Characteristics of AAV Vectors for Cardiomyocytes. It is worth noting that this is the most logical, consistent, and informative part of the review. However, I would like to see more (or any!) numbers or other measurable metrics to characterize vector efficiency and tropism. How broad are “broadened tropism,” “broadest tropism,” and “exceptional tropism”? Why are “enhanced,” “exceptional,” “most,” “highly,” “extremely,” and “superior” vector efficiencies insufficient for CMC transduction and require the development of novel serotypes (Lines 308-310)?
7. Conclusions and Perspectives. I don't know which section Fig. 2 should belong to, but it is not mentioned anywhere and, apparently, is not needed by the authors for their narrative.

Reviewer 3 Report

Comments and Suggestions for Authors

In the review article 'Gene Therapy for Heart Failure: Impact on Mitochondrial Dysfunction' the authors have summarized the topic very well. However, I have some points, which can be improved before publication.

1.) Please define the term heart failure in more detail? Do you mean ischemic cardiomyopathy or dilated cardiomyopathy or HCM or RCM? This term is too broad and should be specified.

2.) Figure 1 is unsharp and the size should be increased. The figure legend should be expanded. Please explain all relevant points of this figure.

3.) Table 1. Please use the official nomenclature and the official gene names. hß3AR, hNdufs6 are not the official gene names.

4.) When you mention a human gene name the first time, I suggest to add an OMIM identifier in brackets.

5.) Figure. Please increase the size. Was this figure generated with Biorender? Then you have to mention this within the Figure legends.

6.) I would add also a short paragraph explaining the genetic etiology of different cardiomyopathies leading to heart failure and the mitochondrial defects in genetic cardiomyopathies. For example, it is known that pathogenic mutations in the DES gene, encoding the structural intermediate filament protein desmin, cause defects of the mitochondria (see 'Desminopathy: Novel Desmin Variants, a New Cardiac Phenotype, and Further Evidence for Secondary Mitochondrial Dysfunction’).

In summary, I suggest a major revision.

Comments on the Quality of English Language

Can be improved.