Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo

Probably not a substantial claim. The paper’s conclusions may not depend on it.

There was considerable variation in the transgene intensities between replicates, particularly in the C57BL/6J(mtC57BL/6J) control samples.

“However, we observed considerable variation in the transgene intensities between replicates, particularly in the C57BL/6J(mtC57BL/6J) control samples.”

It may state two separate claims as one.

The variants in the endogenous peptides were not detected by mass spectrometry, presumably because of inadequate ionization in the assay, so their relative quantitation in the ATP synthase complex was not possible.

“Unfortunately, the variants in the endogenous peptides were not detected by mass spectrometry, presumably because of inadequate ionization in the assay. Therefore, their relative quantitation in the ATP synthase complex was not possible.”

It may state two separate claims as one.

FLAG-tagged oATP8 protein was detected in whole-cell lysates of brain, liver, skeletal muscle, kidney, and spleen in both male and female 12-week-old C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) mice, but not in age-matched littermate non-transgenic animals.

“The expression of FLAG-tagged oATP8 protein was readily detected in whole-cell lysates of the brain, liver, skeletal muscle, kidney, and spleen in both male and female 12-week-old C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) mice, but not in age-matched littermate non-transgenic animals (Figures 3A and 3C).”

It may state two separate claims as one.

About half the progeny were born transgenic: 48.2% ± 4.3% (SEM) in C57BL/6J(mtC57BL/6J) and 49.8% ± 5.7% (SEM) in C57BL/6J(mtFVB).

“About half the progeny, 48.2% ± 4.3% (SEM) and 49.8% ± 5.7% (SEM) in the C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB), respectively, were born transgenic.”

It may state two separate claims as one.

Next-generation sequencing revealed that a single copy of the oATP8 transgene was correctly inserted in the ROSA26 locus (NC_000072.7::113053020), with 7 pair-end reads spanning the insertion site and a confidence score of 96.

“Next-generation sequencing analysis revealed that a single copy of the oATP8 transgene was correctly inserted in the ROSA26 locus (NC_000072.7::113053020) (7 pair-end reads spanning the insertion site were identified with a confidence score of 96).”

It may state two separate claims as one.

The presence of the transgene did not significantly change anxiety levels, exploratory behavior, locomotion, systemic metabolic changes, feeding and drinking behavior, or exercise capacity and tolerance.

“Our results show that the presence of the transgene did not significantly change any of the aforementioned parameters (Figure 8A; Tables S3–S9).”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Elevated levels of proteins Maoa, Acss3, and Dhodh were observed in male C57BL/6J(mtFVB) non-transgenic mice compared with their transgenic cohorts.

“Elevated levels of proteins Maoa, Acss3, and Dhodh were observed in male C57BL/6J(mtFVB) non-transgenic mice compared with their transgenic cohorts.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Actb, Coro1a, Lcp1, Oxct1, and Pkm were all upregulated in female C57BL/6J(mtFVB) non-transgenic mice compared with female mutant transgenic mice.

“In addition, the proteins Actb, Coro1a, Lcp1, Oxct1, and Pkm were all upregulated in female C57BL/6J(mtFVB) non-transgenic mice compared with female mutant transgenic mice (Figure S4iv).”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Actb was upregulated in female C57BL/6J(mtC57BL/6J) non-transgenic mice compared with female transgenic mice.

“Actb was upregulated in female C57BL/6J(mtC57BL/6J) non-transgenic mice compared with female transgenic mice (Figure S4ii).”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

In our hands, some reported behavior phenotypes associated with the ATP8 polymorphism, such as anxiety, could not be recapitulated.

“In our hands, some of the reported behavior phenotypes associated with this polymorphism, such as anxiety, could not be recapitulated.”

It may state two separate claims as one.

Maximal ATP hydrolysis activity (Vmax) and Km (ATP) of the ATP synthase were not significantly changed between the transgenic and non-transgenic groups in isolated liver mitochondria.

“Maximal ATP hydrolysis activity (Vmax) and Km (ATP) of the ATP synthase were not significantly changed between the transgenic and non-transgenic groups in isolated liver mitochondria (Figure 7B; Table S1).”

It may state two separate claims as one.

PCR analysis showed that successful recombination occurred in both transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) strains, but not in littermate non-transgenic controls.

“PCR analysis of tail DNA using specific primer sets corresponding to the regions spanning the ROSA26 locus and oATP8 transgene showed that successful recombination occurred in both the transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) strains, but not in the littermate non-transgenic controls (Figures 2A and S1B).”

It may state two separate claims as one.

No statistical significance was observed in the wild-type transgenic mitochondria despite appreciable transgene expression across samples.

“We did not observe statistical significance in the wild-type transgenic mitochondria despite appreciable transgene expression across samples.”

It may state two separate claims as one.

All animals generated using the breeding scheme were viable and fertile and born at the expected Mendelian ratio without apparent abnormalities in adult mice.

“All animals generated using the breeding scheme in Figure 1C were viable and fertile and born at the expected Mendelian ratio without apparent abnormalities in adult mice.”

It may state two separate claims as one.

The C57BL/6J(mtFVB) strain harbors the FVB/NJ polymorphism, verified by Sanger sequencing.

“We verified that the C57BL/6J(mtFVB) strain harbors the FVB/NJ polymorphism using Sanger sequencing (Figure S1A).”

It may state two separate claims as one.

In the C57BL/6J(mtC57BL/6J) strain, the apparent levels of transgenic and endogenous ATP8 were comparable and did not show significant differences.

“In contrast, in the C57BL/6J(mtC57BL/6J) strain, the apparent levels of transgenic and endogenous ATP8 were comparable and did not show significant differences.”

It may state two separate claims as one.

All the mice produced were viable, fertile, and born at the expected Mendelian ratio without apparent abnormalities in adult mice.

“All the mice produced were viable, fertile, and born at the expected Mendelian ratio without apparent abnormalities in adult mice.”

It may state two separate claims as one.

The transgene and the exogenous oATP8 protein do not elicit an inflammatory host immune response.

“We also observed that the transgene and the exogenous oATP8 protein do not elicit an inflammatory host immune response.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The sample level variations could be due to selective pressures such as transcriptomic changes surrounding the transgene.

“The sample level variations could be due to selective pressures such as transcriptomic changes surrounding the transgene, although we observed appreciable transgene expression in all samples tested.”

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Bilateral injection of lenadogene nolparvovec in the eyes of LHON patients carrying the m.11778G>A MT-ND4 mutation improved visual acuity and has shown an excellent safety profile.

“Bilateral injection of lenadogene nolparvovec (an AAV-engineered allotopic ND4 gene) in the eyes of LHON patients carrying the m.11778G>A MT-ND4 mutation improved visual acuity and has shown excellent safety profile.41”

• cites [41] Newman, 2023, Randomized trial of bilateral gene therapy injection for m.11778G>A MT-ND4 Leber optic neuropathy, Brain sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The mROSA26 locus was originally identified in mouse ESCs for stable expression of promoter-less transgenes using lentivirus knockin screens.

“The mROSA26 locus was originally identified in mouse ESCs for stable expression of promoter-less transgenes using lentivirus knockin screens51”

• cites [51] Friedrich, 1991, Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice, Genes Dev. sourceCheck pending

It may state two separate claims as one.

Attaching a long MTS from Zea mays 4-hydroxybenzoate polyprenyltransferase seemed beneficial in targeting the human allotopic ATP6 construct and showed OXPHOS function recovery in Leigh's model ATP6 8993T>G mutant cybrids.

“Particularly, attaching a long MTS such as the one from Zea mays mitochondrial inner membrane protein 4-hydroxybenzoate polyprenyltransferase (116 amino acids) seemed beneficial in targeting the human allotopic ATP6 construct and showed OXPHOS function recovery in Leigh’s model ATP6 8993T>G mutant cybrids.43”

• cites [43] Chin, 2018, Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6, Cell Rep. sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it.

In humans, the region surrounding mROSA26 is also surrounded by proto-oncogenes that could influence transgene expression.

“In humans, this region is also surrounded by proto-oncogenes that could influence transgene expression.52”

• cites [52] Sadelain, 2011, Safe harbours for the integration of new DNA in the human genome, Nat. Rev. Cancer sourceNot checked — no full text of the source

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Nagley et al. were the first to implement allotopic expression for mtDNA proteins by recoding the yeast ATP8 gene for the nuclear DNA code, and their work demonstrated successful restoration of respiratory growth in ATP8-null cells.

“Nagley et al. were the first to implement allotopic expression for mtDNA proteins by recoding the yeast ATP8 gene for the nuclear DNA code. Their work demonstrated successful restoration of respiratory growth in ATP8-null cells.39”

• cites [39] Nagley, 1988, Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source

Probably not a substantial claim.

Previous approaches have predominantly used recombinant adenoviruses to target specific organs, such as the eye, expressing allotopic ND4, ATP6, or ND1 transgenes.

“Previous approaches have predominantly used recombinant adenoviruses to target specific organs, such as the eye, expressing allotopic ND4,23 ATP6,24 or ND150 transgenes.”

• cites [23] Yu, 2012, Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice, Mol. Vis.Not checked yet
• cites [24] Dunn, 2012, Nuclear expression of a mitochondrial DNA gene: mitochondrial targeting of allotopically expressed mutant ATP6 in transgenic mice, J. Biomed. Biotechnol. sourceNot checked yet
• cites [50] Liu, 2022, Gene therapy restores mitochondrial function and protects retinal ganglion cells in optic neuropathy induced by a mito-targeted mutant ND1 gene, Gene Ther. sourceCheck pending

Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Densitometry analysis of western blots is a semiquantitative technique, and determining the exact ratios of oATP8/ATP8 warrants further analysis using alternative methods.

“It is important to emphasize that densitometry analysis of western blots is a semiquantitative technique and determining the exact ratios of oATP8/ATP8 warrants further analysis using alternative methods.”

Probably not a substantial claim. The paper may give no reason why it holds.

The FVB/NJmtFVB/NJ mouse strain is the only available model harboring a spontaneous single-nucleotide polymorphism in the mitochondrial ATP8 gene.

“The FVB/NJmtFVB/NJ mouse (JAX strain no. 001800) strain is the only available model harboring a spontaneous single-nucleotide polymorphism in the mitochondrial ATP8 gene.”

It may state two separate claims as one.

Allotopic expression of ATP6 in patient cybrids with specific mutations such as mt.8993T>G or mt.8529G>A shows promise and can restore partial function.

“Allotopic expression of ATP6 in patient cybrids with specific mutations such as the mt.8993T>G or (mt.8529G>A) shows promise and can restore partial function.27,29,40”

• cites [27] Boominathan, 2016, Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant, Nucleic Acids Res. sourceCheck pending
• cites [29] Manfredi, 2002, Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus, Nat. Genet. sourceNot checked — no full text of the source
• cites [40] Zullo, 2005, Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy, Rejuvenation Res. sourceNot checked — no full text of the source

It may state two separate claims as one.

The similar activity of ATP synthase with transgene to non-transgenic control suggests successful integration and function.

“The ATP synthase with transgene had similar activity to non-transgenic control, suggesting successful integration and function.”

It may state two separate claims as one.

The exogenous oATP8 gene is constitutively expressed, translated, and targeted in vivo into the correct mitochondrial complex in all tissues checked.

“The following data demonstrate that the exogenous oATP8 gene is constitutively expressed, translated, and targeted in vivo into the correct mitochondrial complex in all the tissues we checked.”

It may state two separate claims as one.

Successful allotopic expression of mtDNA-encoded genes requires optimization of coding and non-coding elements such as mandatory changes to the gene sequence to synchronize nuclear-cytosolic translation, appending a suitable MTS, and appropriate 5′ and 3′ UTR sequences.

“Successful allotopic expression of mtDNA-encoded genes requires optimization of important coding and non-coding elements such as (1) mandatory changes to the gene sequence to synchronize nuclear-cytosolic translation, (2) appending a suitable MTS to target the protein to the mitochondria, and (3) appropriate 5′ and 3′ UTR sequences in regulating transcription and translation.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The organ-level variation in transgene expression could be attributed to varying demands for mitochondrial biogenesis and function.

“The organ-level variation in transgene expression could be attributed to varying demands for mitochondrial biogenesis and function.”

• AssumptionRests on an assumption: Tissues such as the heart and brain rely more on oxidative phosphorylation for their energy demands, whereas the spleen is less so.

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

Challenges to successful allotopic expression include engineering and nuclear integration of an optimized construct, targeting an exogenous cytosolic protein to the mitochondria, and functional assembly and rescue of a mutant mitochondrial phenotype.

“Challenges to successful allotopic expression have been described previously, including (1) the engineering and nuclear integration of an optimized construct to express from the nucleus, (2) the targeting of an exogenous cytosolic protein to the mitochondria, and (3) the functional assembly and rescue of a mutant mitochondrial phenotype.32”

• cites [32] Saravanan, 2022, The Mitochondrial Genome in Aging and Disease and the Future of Mitochondrial Therapeutics, Biomedicines sourceNot checked yet

Probably not a substantial claim. The paper’s conclusions may not depend on it.

All lines used in the studies were in the C57BL/6J nuclear background strain.

“All lines used in the studies were in the C57BL/6J nuclear background strain.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

All behavior experiments were conducted at the same time between 8 a.m. and 1 p.m. during the light phase in a dimly lit room.

“All behavior experiments were conducted at the same time between 8 a.m. and 1 p.m. during the light phase in a dimly lit room.”

Probably not a substantial claim. It may be too vague to check against evidence. The paper may give no reason why it holds.

The development of this gene therapy technology in vivo provides the opportunity to demonstrate the rescue of whole-body function in models that exhibit pathological mtDNA mutations.

“The development of this gene therapy technology in vivo provides the opportunity to demonstrate the rescue of whole-body function in models that exhibit pathological mtDNA mutations.”

• AssumptionRests on an assumption: The pathological effects of mtDNA mutations that arise with age extend beyond localized regions.

Probably not a substantial claim.

Previously, only a fraction of allotopically expressed mitochondrial protein integrates into the proper mitochondrial complex.

“Previously, we have shown how only a fraction of allotopically expressed mitochondrial protein integrates into the proper mitochondrial complex.27,33”

• cites [27] Boominathan, 2016, Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant, Nucleic Acids Res. sourceCheck pending
• cites [33] Lewis, 2020, Codon optimization is an essential parameter for the efficient allotopic expression of mtDNA genes, Redox Biol. sourceNot checked yet

Probably not a substantial claim.

We prepared purified mtDNA from conplastic animals to verify for homoplasmy of the mitochondrial 7778 G-T mutation in the ATP8 gene.

“We prepared purified mtDNA from conplastic animals to verify for homoplasmy of the mitochondrial 7778 G-T mutation in the ATP8 gene.”

Probably not a substantial claim.

To verify for construct insertion in the ROSA26 locus, we conducted next-generation sequencing.

“To verify for construct insertion in the ROSA26 locus, we conducted next-generation sequencing.”

It may state two separate claims as one.

Allotopic gene therapy for the ATP8 gene is feasible and safe to use in vivo.

“The absence of detectable differences between transgenic and non-transgenic animals highlights that allotopic gene therapy for the ATP8 gene is feasible and safe to use in vivo.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

For all mice, testing was performed between 8 a.m. and 1 p.m. to minimize the influence of the time of day.

“For all mice, testing was performed between 8 a.m. and 1 p.m. to minimize the influence of the time of day.”

It may state two separate claims as one.

We utilized the conplastic C57BL/6J(mtFVB) mouse strain with a naturally occurring ATP8 missense mutation that exhibits mild phenotypes not significantly different from C57BL/6J(mtC57BL/6J) animals.

“In our study, we utilized the conplastic C57BL/6J(mtFVB) mouse strain with a naturally occurring ATP8 missense mutation that exhibits mild phenotypes not significantly different from C57BL/6J(mtC57BL/6J) animals.31”

• cites [31] Yu, 2009, Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains, Genome Res. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

All experiments involving the use of mice were reviewed and approved by the IACUC of the Buck Institute and conducted with strict adherence to NIH guidelines under protocol A10226.

“All experiments involving the use of mice were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Buck Institute and conducted with strict adherence to the National Institutes of Health Animal Care and Guidelines under protocol A10226.”

Probably not a substantial claim.

Enhanced protein stability of transgenic ATP8 in the FVB strain due to differences in the aspartic acid substitution at the fifth position might explain the integration difference.

“This might be attributed to enhanced protein stability of transgenic ATP8 in the FVB strain due to differences in the aspartic acid substitution at the fifth position.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Analysis of the open-field data was completed blind to the experimental groups.

“Analysis of the open-field data was completed blind to the experimental groups.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

We carefully titrated the FCCP exposure to the mitochondria to ensure maximal respiratory capacity was captured.

“We also carefully titrated the FCCP exposure to the mitochondria to ensure maximal respiratory capacity was captured.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Two animals that slipped off the platform were excluded from the analysis.

“Two animals that slipped off the platform were excluded from the analysis.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The transgenic and non-transgenic animals used were littermates.

“The transgenic and non-transgenic animals used were littermates.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

We titrated the total amount of mitochondrial protein plated to ensure that the linear range of mitochondrial response to stimuli was within the dynamic range of the instrument.

“Prior to running the assay, we titrated the total amount of mitochondrial protein plated to ensure that the linear range of mitochondrial response to stimuli was within the dynamic range of the instrument.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The IC50 of oligomycin at the F1FO complex was calculated by titrating the oligomycin concentration.

“The IC50 of oligomycin at the F1FO complex was calculated by titrating the oligomycin concentration.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

In all the studies, efforts were made to use equal numbers of male and female mice within each experimental group.

“In all the studies, efforts were made to use equal numbers of male and female mice within each experimental group.”

Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.

The applicability of this approach to other mtDNA genes may require a case-by-case validation, considering factors such as protein length, number of transmembrane domains, and the efficiency of import and targeting to their respective OXPHOS complexes.

“The applicability of this approach to other mtDNA genes may require a case-by-case validation, considering factors such as protein length, number of transmembrane domains, and the efficiency of import and targeting to their respective OXPHOS complexes.”

• AssumptionRests on an assumption: ATP8 is a small protein (∼8 kDa) in the mitochondrial genome with a single transmembrane domain.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

To determine the optimal alamethicin concentration, we titrated alamethicin in an NADH-driven respiration experiment.

“To determine the optimal alamethicin concentration, we titrated alamethicin in an NADH-driven respiration experiment in the XFe96 Agilent Seahorse Analyzer, as described previously.68”

• cites [68] Divakaruni, 2018, In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents, Anal. Biochem. sourceCheck pending

It may state two separate claims as one.

The expression of the allotopic ATP8 protein is well tolerated and does not result in adverse proteostasis.

“Mitochondrial proteome analysis for liver mitochondria purified from transgenic and non-transgenic mice did not show any discernible changes indicating that the expression of the allotopic ATP8 protein is well tolerated and does not result in adverse proteostasis.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A no-reverse transcriptase control was included in each reaction set to rule out non-specific priming.

“A no-reverse transcriptase control was included in each reaction set to rule out non-specific priming.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Behavior in the elevated plus maze was recorded using a video camera and later analyzed by a blinded observer.

“Their behavior was recorded using a video camera and later analyzed by a blinded observer.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Proton production rates were calculated by fitting to a pH standard curve generated by serial injections of HCl.

“Proton production rates were calculated by fitting to a pH standard curve generated by serial injections of HCl from the injection ports, as described previously.69”

• cites [69] Mookerjee, 2015, Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate, J. Vis. Exp. sourceCheck pending

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The first 24 h of metabolic cage data were excluded from analysis because the animals were acclimating to the metabolic cage.

“We excluded the first 24 h as the animals were acclimating to the metabolic cage.”

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Construct insertion into the ROSA26 locus was further ascertained by Sanger sequencing using proprietary primers specific to the ROSA26 locus.

“Construct insertion into the ROSA26 locus was further ascertained by Sanger sequencing using the proprietary primers specific to the ROSA26 locus provided by Applied Stem Cell (Milipitas, CA).”