One episode of low intensity aerobic exercise prior to systemic AAV9 administration augments transgene delivery to the heart and skeletal muscle.

INTRODUCTION: The promising potential of adeno-associated virus (AAV) gene delivery strategies to treat genetic disorders continues to grow with an additional three AAV-based therapies recently approved by the Food and Drug Administration and dozens of others currently under evaluation in clinical trials. With these developments, it has become increasingly apparent that the high doses currently needed for efficacy carry risks of toxicity and entail enormous manufacturing costs, especially for clinical grade products. Strategies to increase the therapeutic efficacy of AAV-mediated gene delivery and reduce the minimal effective dose would have a substantial impact on this field. We hypothesized that an exercise-induced redistribution of tissue perfusion in the body to favor specific target organs via acute aerobic exercise prior to systemic intravenous (IV) AAV administration could increase efficacy. BACKGROUND: Aerobic exercise triggers an array of downstream physiological effects including increased perfusion of heart and skeletal muscle, which we expected could enhance AAV transduction. Prior preclinical studies have shown promising results for a gene therapy approach to treat Barth syndrome (BTHS), a rare monogenic cardioskeletal myopathy, and clinical studies have shown the benefit of low intensity exercise in these patients, making this a suitable disease in which to test the ability of aerobic exercise to enhance AAV transduction. METHODS: Wild-type (WT) and BTHS mice were either systemically administered AAV9 or completed one episode of low intensity treadmill exercise immediately prior to systemic administration of AAV9. RESULTS: We demonstrate that a single episode of acute low intensity aerobic exercise immediately prior to IV AAV9 administration improves marker transgene delivery in WT mice as compared to mice injected without the exercise pre-treatment. In BTHS mice, prior exercise improved transgene delivery and additionally increased improvement in mitochondrial gene transcription levels and mitochondrial function in the heart and gastrocnemius muscles as compared to mice treated without exercise. CONCLUSIONS: Our findings suggest that one episode of acute low intensity aerobic exercise improves AAV9 transduction of heart and skeletal muscle. This low-risk, cost effective intervention could be implemented in clinical trials of individuals with inherited cardioskeletal disease as a potential means of improving patient safety for human gene therapy.

Matrix produced by diseased cardiac fibroblasts affects early myotube formation and function.

The extracellular matrix (ECM) provides both physical and chemical cues that dictate cell function and contribute to muscle maintenance. Muscle cells require efficient mitochondria to satisfy their high energy demand, however, the role the ECM plays in moderating mitochondrial function is not clear. We hypothesized that the ECM produced by stromal cells with mitochondrial dysfunction (Barth syndrome, BTHS) provides cues that contribute to metabolic dysfunction independent of muscle cell health. To test this, we harnessed the ECM production capabilities of human pluripotent stem-cell-derived cardiac fibroblasts (hPSC-CFs) from healthy and BTHS patients to fabricate cell-derived matrices (CDMs) with controlled topography, though we found that matrix composition from healthy versus diseased cells influenced myotube formation independent of alignment cues. To further investigate the effects of matrix composition, we then examined the influence of healthy- and BTHS-derived CDMs on myotube formation and metabolic function. We found that BTHS CDMs induced lower fusion index, lower ATP production, lower mitochondrial membrane potential, and higher ROS generation than the healthy CDMs. These findings imply that BTHS-derived ECM alone contributes to myocyte dysfunction in otherwise healthy cells. Finally, to investigate potential mechanisms, we defined the composition of CDMs produced by hPSC-CFs from healthy and BTHS patients using mass spectrometry and identified 15 ECM and related proteins that were differentially expressed in the BTHS-CDM compared to healthy CDM. Our results highlight that ECM composition affects skeletal muscle formation and metabolic efficiency in otherwise healthy cells, and our methods to generate patient-specific CDMs are a useful tool to investigate the influence of the ECM on disease progression and to investigate variability among diseased patients. STATEMENT OF SIGNIFICANCE: Muscle function requires both efficient metabolism to generate force and structured extracellular matrix (ECM) to transmit force, and we sought to examine the interactions between metabolism and ECM when metabolic disease is present. We fabricated patient-specific cell derived matrices (CDMs) with controlled topographic features to replicate the composition of healthy and mitochondrial-diseased (Barth syndrome) ECM. We found that disease-derived ECM negatively affects metabolic function of otherwise healthy myoblasts, and we identified several proteins in disease-derived ECM that may be mediating this dysfunction. We anticipate that our patient-specific CDM system could be fabricated with other topographies and cell types to study cell functions and diseases of interest beyond mitochondrial dysfunction and, eventually, be applied toward personalized medicine.

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts.

A splice site mutation in the canine pyruvate dehydrogenase kinase 4 (PDK4) gene has been shown to be associated with the development of dilated cardiomyopathy (DCM) in Doberman Pinchers (DPs). Subsequent studies have successfully demonstrated the use of dermal fibroblasts isolated from DPs as models for PDK4 deficiency and have shown activation of the intrinsic (mitochondrial mediated) apoptosis pathway in these cells under starvation conditions. For this study, we sought to further explore the functional consequences of PDK4 deficiency in DP fibroblasts representing PDK4wt/wt, PDK4wt/del, and PDK4del/del genotypes. Our results show that starvation conditions cause increased perinuclear localization of mitochondria and decreased cell proliferation, altered expression levels of pyruvate dehydrogenase phosphatase (PDP) and pyruvate dehydrogenase (PDH), dramatically increased PDH activity, and an impaired response to mitochondrial stress in affected cells. In sum, these results show the broad impact of PDK4 deficiency and reveal mechanistic pathways used by these cells in an attempt to compensate for the condition. Our data help to elucidate the mechanisms at play in this extremely prevalent DP disorder and provide further support demonstrating the general importance of metabolic flexibility in cell health.

Increased mtDNA Abundance and Improved Function in Human Barth Syndrome Patient Fibroblasts Following AAV-TAZ Gene Delivery.

Barth syndrome (BTHS) is a rare, X-linked, mitochondrial disorder caused by mutations in the gene encoding tafazzin. BTHS results in cardiomyopathy, muscle fatigue, and neutropenia in patients. Tafazzin is responsible for remodeling cardiolipin, a key structural lipid of the inner mitochondrial membrane. As symptoms can vary in severity amongst BTHS patients, we sought to compare mtDNA copy numbers, mitochondrial fragmentation, and functional parameters between primary dermal BTHS fibroblasts isolated from patients with two different mutations in the TAZ locus. To confirm cause‒effect relationships and further support the development of gene therapy for BTHS, we also characterized the BTHS cells following adeno-associated virus (AAV)-TAZ transduction. Our data show that, in response to AAV-TAZ transduction, these remarkably dynamic organelles show recovery of mtDNA copy numbers, mitochondrial structure, and mitochondrial function, providing additional evidence to support the therapeutic potential of AAV-mediated gene delivery for BTHS. This study also demonstrates the direct relationship between healthy mitochondrial membrane structure and maintenance of proper levels of mtDNA copy numbers.

AAV9-TAZ Gene Replacement Ameliorates Cardiac TMT Proteomic Profiles in a Mouse Model of Barth Syndrome.

Barth syndrome (BTHS) is a rare mitochondrial disease that causes severe cardiomyopathy and has no disease-modifying therapy. It is caused by recessive mutations in the gene tafazzin (TAZ), which encodes tafazzin-an acyltransferase that remodels the inner mitochondrial membrane lipid cardiolipin. To identify novel mechanistic pathways involved in BTHS and evaluate the effects of gene therapy on proteomic profiles, we performed a multiplex tandem mass tagging (TMT) quantitative proteomics analysis to compare protein expression profiles from heart lysates isolated from BTHS, healthy wild-type (WT), and BTHS treated with adeno-associated virus serotype 9 (AAV9)-TAZ gene replacement as neonates or adults. 197 proteins with ≥2 unique peptides were identified. Of these, 91 proteins were significantly differentially expressed in BTHS compared to WT controls. Cause-effect relationships between tafazzin deficiency and altered protein profiles were confirmed through demonstrated significant improvements in expression levels following administration of AAV9-TAZ. The importance of TMEM65 in Cx43 localization to cardiac intercalated discs was revealed as a novel consequence of tafazzin deficiency that was improved following gene therapy. This study identifies novel mechanistic pathways involved in the pathophysiology of BTHS, demonstrates the ability of gene delivery to improve protein expression profiles, and provides support for clinical translation of AAV9-TAZ gene therapy.

AAV-Mediated TAZ Gene Replacement Restores Mitochondrial and Cardioskeletal Function in Barth Syndrome.

Barth syndrome (BTHS) is a rare mitochondrial disease that affects heart and skeletal muscle and has no curative treatment. It is caused by recessive mutations in the X-linked gene TAZ, which encodes tafazzin. To develop a clinically relevant gene therapy to restore tafazzin function and treat BTHS, three different adeno-associated virus serotype 9 vectors were tested and compared to identify the optimal promoter-cytomegalovirus (CMV), desmin (Des), or a native tafazzin promoter (Taz)-for TAZ expression following intravenous administration of 1 × 1013 vector genomes/kilogram to a mouse model of BTHS as either neonates (1-2 days of age) or adults (3 months of age). At 5 months of age, evaluations of biodistribution and TAZ expression levels, mouse activity assessments, fatigue in response to exercise, muscle strength, cardiac function, mitochondrial structure, oxygen consumption, and electron transport chain complex activity assays were performed to measure the extent of improvement in treated mice. Each promoter was scored for significant improvement over untreated control mice and significant improvement compared with the other two promoters for every measurement and within each age of administration. All three of the promoters resulted in significant improvements in a majority of the assessments compared with untreated BTHS controls. When scored for overall effectiveness as a gene therapy, the Des promoter was found to provide improvement in the most assessments, followed by the CMV promoter, and finally Taz regardless of injection age. This study provides substantial support for translation of an adeno-associated virus serotype 9-mediated TAZ gene replacement strategy using a Des promoter for human BTHS patients in the clinic.

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma.

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast-cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow-cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose-deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7-related metabolic pathways is a viable therapeutic strategy.

Impact of PYROXD1 deficiency on cellular respiration and correlations with genetic analyses of limb-girdle muscular dystrophy in Saudi Arabia and Sudan.

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.

PDK4 Deficiency Induces Intrinsic Apoptosis in Response to Starvation in Fibroblasts from Doberman Pinschers with Dilated Cardiomyopathy.

The Doberman pinscher (DP) canine breed displays a high incidence of idiopathic, nonischemic dilated cardiomyopathy (DCM) with increased mortality. A common mutation in DPs is a splice site deletion in the pyruvate dehydrogenase kinase 4 (PDK4) gene that shows a positive correlation with DCM development. PDK4, a vital mitochondrial protein, controls the switch between glycolysis and oxidative phosphorylation based upon nutrient availability. It is likely, although unproven, that DPs with the PDK4 mutation are unable to switch to oxidative phosphorylation during periods of low nutrient availability, and thus are highly susceptible to mitochondrial-mediated apoptosis. This study investigated cell viability, mitochondrial stress, and activation of the intrinsic (mitochondrial mediated) apoptotic pathway in dermal fibroblasts from DPs that were healthy (PDK4wt/wt), heterozygous (PDK4wt/del), and homozygous (PDK4del/del) for the PDK4 mutation under conditions of high (unstarved) and low (starved) nutrient availability in vitro. As hypothesized, PDK4wt/del and PDK4del/del cells showed evidence of mitochondrial stress and activation of the intrinsic apoptotic pathway following starvation, while the PDK4wt/wt cells remained healthy and viable under these conditions. Adeno-associated virus (AAV) PDK4-mediated gene replacement experiments confirmed cause-effect relationships between PDK4 deficiency and apoptosis activation. The restoration of function observed following administration of AAV-PDK4 provides strong support for the translation of this gene therapy approach into the clinical realm for PDK4-affected Dobermans.

Heterologous expression of mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) from Aspergillus fumigatus rescues impaired growth in Δndt1Δndt2 Saccharomyces cerevisiae strain.

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD+), generating a large mitochondrial membrane potential. The NAD+ carrier utilized the electrochemical proton gradient to drive NAD+ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.