26th Annual Symposium of the United Mitochondrial Disease Foundation -Mitochondrial Medicine 2023 – Abstracts

Abstract #	Abstract Title	Presenter Name
2023PA-0000000113	Progress Towards Novel Target-Based Small Molecule Therapeutics for Pyruvate Dehydrogenase Complex Deficiency due to Specific Recurrent E1α Amino Acid Replacements	Jirair	Bedoyan
2023PA-0000000114	DELE1 oligomers revealed by cryo-EM structure promote the integrated stress response activation	Jie	Yang
2023PA-0000000116	Outpatient Arginine Infusions as a Preventive Treatment for Stroke-Like Episodes in MELAS Syndrome: Early Results and Family Experience.	Heather	Pomella
2023PA-0000000117	PPARδ AGONIST MAVODELPAR (REN001) Improves Mitochondrial Function in Skeletal Muscle: A Potential Treatment for Primary Mitochondrial Myopathies	John W.	Adams
2023PA-0000000118	Mavodelpar Clinical Development Program In Adult Patients With Primary Mitochondrial Myopathy (Pmm): Results From Phase 1b Study And Design Of Ongoing Pivotal Study (Stride)	Gráinne	Gorman
2023PA-0000000119	Importance Of Patient and Caregiver Voice For Living With Primary Mitochondrial Myopathies And Associated Multi-Organ Involvement	Margaret	Moore
2023PA-0000000120	Peripheral macrophages causally contribute to disease onset and progression in the Ndufs4(KO) model of Leigh syndrome	Allison	Hanaford
2023PA-0000000121	Quantification of Mitochondrial Heteroplasmy in Human Heart	Mahitha	Roy
2023PA-0000000122	Neuron-specific CASK Loss in Mice Causes Epileptic Encephalopathy Associated with Impaired Cerebral Cortex Development and Mitochondrial Dysfunction	Hemangi	Bhonsle
2023PA-0000000123	Inflammatory And Interferon Gene Expression Signatures in Patients with Mitochondrial Disease	Emily	Warren
2023PA-0000000124	A Novel APOO Deletion in An African American Male with Epilepsy, Autism, Ataxia, Hypotonia and Cognitive Deficit	Kumarie	Latchman
2023PA-0000000126	Systemic cytokine storm exacerbates acute influenza A infection in Ndufs4 KO mouse	Amanda	Fuchs
2023PA-0000000128	A Case Study of A Trio of Siblings Utilizing Elamipretide for POLG1 Mitochondrial Disease	Neena	Patel
2023PA-0000000129	Loss of pathogenic mitochondrial tRNA mutations during the development of adaptive immune responses	Joanna	Rorbach
2023PA-0000000130	The mitochondrial phosphatase Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy	Natalie	Niemi
2023PA-0000000131	Non-pathogenic variation in mitochondrial DNA modulates murine SARS-CoV-2 pathogenesis	Yentli	Soto Albrecht
2023PA-0000000133	Remdesivir increases mtdna copy number causing mild alterations to oxidative phosphorylation	Nicole	DeFoor
2023PA-0000000134	Harnessing the potential of transcriptional adaptation as a mechanism for mitochondrial genetic disorders	Adriana	Morales Gomez
2023PA-0000000135	FDA-approved PDE4 inhibitors reduce the dominant toxicity of ALS–FTD-associated CHCHD10S59L in Drosophila and human cells	Nam Chul	Kim
2023PA-0000000136	Emergency Use of doxectine and doxribtiminein an Adult Patient with TK2d Following a Traumatic Fall	Lindsey	Miller
2023PA-0000000137	Hemin Impairs Mitochondrial Gene Expression and Induces Guanine Quadruplexes in Human Renal Proximal Tubule Cells	Ryan	Snyder
2023PA-0000000138	Targeted Down Regulation Of Core Mitochondrial Genes During Sars-Cov-2 Infection	Joseph	Guarnieri
2023PA-0000000140	Validation Of The Mitochondrial Myopathy Function Scale	Jean	Flickinger
2023PA-0000000143	Mitochondrial Mobility Performance Levels	Jean	Flickinger
2023PA-0000000145	Preservation of bioenergetics and inhibition of ferroptosis with the novel compound SBT-588 in Friedreich’s Ataxia cell models	Hatim	Zariwala
2023PA-0000000146	Behavioral Characterization Of MEPAN Mouse Model	Valeria	Carosi
2023PA-0000000147	Expanded Clinical and Neuroradiological Phenotype of RARS2-Related Mitochondrial Disorder	Fernando	Scaglia
2023PA-0000000148	Characterization of mitochondrial augmentation at the single cell level	Noa	Sher
2023PA-0000000149	Targeting MNRR1 as a therapeutic in models of mitochondrial dysfunction.	Siddhesh	Aras
2023PA-0000000150	Colonic Mitochondrial Dysfunction in Rodent Models of PCOS	Kristin	Edwards
2023PA-0000000151	The OMA1-DELE1 mitochondrial integrated stress response is activated by diverse mitochondrial stressors to promote growth and survival in mitochondrial myopathy	Hsin-Pin	Lin
2023PA-0000000152	Development and Validation of a Mitochondrial Disease-Specific Fatigue Questionnaire	Amel	Karaa
2023PA-0000000153	Development of a semi-quantitative mitochondrial protein assay	Christine	Kong
2023PA-0000000155	Elamipretide Demonstrates Target Engagement and Signs of Visual Function Benefits by Protecting Photoreceptors in Dry AMD	Gene	Kelly
2023PA-0000000156	Inpatient Epidemiology, Healthcare Utilization And Comorbidities Of Melas: A National Inpatient Sample Study	Jirat	Chenbhanich
2023PA-0000000157	A deep mutational scanning framework for the clinical and functional characterization of OxPhos proteins	Andrew	Sung
2023PA-0000000160	Single-cell evaluation of bioenergetics by flow cytometry (e-Flo)	Jose Luis	Marin Franco
2023PA-0000000163	In Vitro System For Evaluating The Effect Of Mitochondrial Replacement On T Cell Phenotypes	Jillian	Jetmore
2023PA-0000000164	Mitochondria And Reproductive Biology	Jeffrey	Haltom
2023PA-0000000165	Exploring new strategies for CoQ supplementation	Laura	Steenberge
2023PA-0000000166	Therapeutic modeling in human fibroblast and C. elegans models of C12ORF65 primary mitochondrial disease	Cristina	Remes
2023PA-0000000167	Efficacy and safety of elamipretide in subjects with primary mitochondrial disease resulting from pathogenic nuclear DNA mutations (nPMD): phase 3 study design	Amel	Karaa
2023PA-0000000168	Mitochondrial Dysfunction In Neuropsychiatric Disorders	Katherine	Mitchell
2023PA-0000000170	Patient co-creation and collaboration in thymidine kinase 2 deficiency (TK2d): Incorporating a stakeholder-led project steering committee into a qualitative observational study	Cristy	Balcells
2023PA-0000000171	Mapping mitochondrial disease global registries: A comprehensive review of publicly listed patient-driven and clinical umbrella registries related to mitochondrial disorders	Cristy	Balcells
2023PA-0000000172	Current Status of the Phase 3 Trial of Dichloroacetate (DCA) for Pyruvate Dehydrogenase Complex Deficiency (PDCD)	Peter	Stacpoole
2023PA-0000000173	Association of 37 mitochondrial DNA genes with Primary Mitochondrial Disease: Standardized assessment using the ClinGen Clinical Validity Framework.	Elizabeth	McCormick
2023PA-0000000174	Application of mitochondrial DNA variant interpretation specification guidelines to 96 MITOMAP-Confirmed disease-associated variants: Outcomes and identification of strengths and limitations	Elizabeth	McCormick
2023PA-0000000175	Uveal melanoma cells exhibit variable growth patterns and distinct mitochondrial respiration profile compared with primary human uveal melanocytes.	Piotr	Kopinski
2023PA-0000000176	LACTB deletion alters mitochondrial metabolism and impacts intermembrane contact sites	Gunjan	Purohit
2023PA-0000000177	MSeqDR Virtual Registry of 7,500 Leigh Syndrome and Primary Mitochondrial Disease Cases Constructed through Semi-Automated Literature Mining and Expert Curation	Lishuang	Shen
2023PA-0000000178	FALCON: A Randomized, Placebo-Controlled Study of the Efficacy of KL1333 in Adult Patients with Primary Mitochondrial Disease	Magnus	Hansson
2023PA-0000000179	Identifying Neuroimaging Patterns in Mitochondrial Leukoencephalopathies	Sonal	Sharma
2023PA-0000000180	Characterization of 19 mitochondrial aminoacyl-tRNA synthetases in C. elegans	Cristina	Remes
2023PA-0000000181	Deficits in mitochondrial oxidative phosphorylation enhance SARS-CoV-2 replication	Ryan	Morrow
2023PA-0000000182	High-throughput pre-clinical evaluation of candidate therapies using RNA interference of DLD-based Primary Mitochondrial Disease in Caenorhabditis elegans	Shannon	Schrope
2023PA-0000000183	Implementing the CHOP Mito Care Pathway to deliver clinical care in the inpatient setting	Cassandra	Pantano
2023PA-0000000184	Facilitating community-based genomic data analysis in primary mitochondrial disease: Mitochondrial Disease Sequence Data Resource (MSeqDR) and mitoSHARE patient registry collaboration to support genomic data discoveries.	Emily	Bogush
2023PA-0000000186	Racial Disparity in the Diagnosis of Mitochondrial Disease	Daniel	McGinn
2023PA-0000000187	A Large-Scale Drug Screen For Compounds That Improve Reductive Stress In Melas Cardiomyocytes	Graeme	Preston
2023PA-0000000191	m.12315G>A pathogenic variant in MT-TL2 presenting with a MELAS-like clinical syndrome and depletion of nitric oxide donors	Fernando	Scaglia
2023PA-0000000192	Identification of two mitophagy modulators that rescue mitochondrial stress in a heteroplasmic single large-scale mtDNA deletion (SLSMD) C. elegans animal model.	Suraiya	Haroon
2023PA-0000000193	Cardiac Manifestations of Mitochondrial Disease: A Single Center Experience	Divakar	Mithal
2023PA-0000000194	Modeling exercise intolerance through maximal swimming capacity and whole body oxygen consumption capacity with exercise in adult zebrafish models of mitochondrial disease	Leonard	Burg
2023PA-0000000195	Sex-specific colonic mitochondrial dysfunction and improvement with mitochondrial targeted therapies in the indomethacin-induced inflammatory bowel disease model in rats	Ngoc	Hoang
2023PA-0000000198	Targeting Mitochondrial Metabolism using Nervonic Acid in Adrenoleukodystroph Note: Primary author - Marcia Terluk will be available for questions	Chenxu	Li
2023PA-0000000200	Succinate Does Not Increase Reactive Oxygen Species Generation in Phosphorylating Human Mitochondria	Irene	Yee
2023PA-0000000201	Development Of Osteosarcoma Xenografts In Zebrafish Models Of Mitochondrial Disease	Nicole A.	Woodard
2023PA-0000000202	Biomarker Cardiolipin Ratios Predict Phenotypic Responders in Barth Syndrome Patients with Cardiomyopathy: Analysis from TAZPOWER OLE 168 Week Study	John	Campbell
2023PA-0000000203	TFAM C-terminal Tail is Dispensable for mtDNA Transcription and Replication	Mikhail	Alexeyev
2023PA-0000000204	A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication	Mikhail	Alexeyev
2023PA-0000000205	TFAM's Contributions to mtDNA Replication and OXPHOS Biogenesis are Genetically Separable	Mikhail	Alexeyev
2023PA-0000000206	Single Large-Scale Mitochondrial DNA Deletion Syndromes (SLSMDS) Overlap with Rothmund-Thomson Syndrome (RTS): Two New Cases and a Review of the Literature	Amy	Goldstein
2023PA-0000000207	Navigating the Mysteries of mtDNA Disease: Collaborative Patient-Specialist Communication in Clinical Trials	Andrea	Hoffmeier
2023PA-0000000208	Dengue Virus Triggers Inflammation by Disrupting Host Mitochondrial Quality Control and Homeostasis	Gulam	Syed
2023PA-0000000209	Clinical Manifestations and Disease Burden of Primary Mitochondrial Myopathies (PMM): Results from a Patient Journey Analysis Shows Substantial Healthcare Resource Utilization	Mai	Sirimanne
2023PA-0000000210	From Clinical Manifestations of Primary Mitochondrial Myopathies (PMM) to Diagnosis: Results from a Patient Journey Analysis Shows Limited Utilization of Genetic Testing	Mai	Sirimanne
2023PA-0000000211	Results from a Phase 2a study evaluating zagociguat, a CNS-penetrant sGC stimulator, in adults with Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like Episodes (MELAS)	Chad	Glasser
2023PA-0000000213	Characterization of a Novel UQCRC1 Variant in a Patient with Progressive Weakness, Pain and Sleep Issues Reveal a Functional Mitochondrial Defect	Gerardo	Piroli
2023PA-0000000214	Enhanced Mitochondrial Base Editing System for Near Complete Mitochondrial Genome Editing in Human Primary Cells Suitable for Disease Modeling and for Potential Therapeutic Applications	Bibekananda	Kar
2023PA-0000000215	Fishing for Cures: Zebrafish as a Pioneering In Vivo Model to Help Solve Mitochondrial Medicine Odysseys	Ankit	Sabharwal
2023PA-0000000216	The phenotypic severity of overlapping mitochondrial deletions is positively associated with heteroplasmy level in two individuals	Lana	Sheta
2023PA-0000000218	Linking altered microglial metabolism to an impaired inflammatory response in Leigh syndrome	Norma	Frizzell
2023PA-0000000219	Using Creighton Model FertilityCare™System Biomarkers to Evaluate and Treat Hormone Dysfunction in Inflammatory Disease, Endometriosis, with Case Study	Suzanne	Scheller
2023PA-0000000221	Mitochondrial Trans-2-Enoyl Coenzyme A Reductase (Mecr) Regulates CD4+ T Cell Function	Kaylee	Steiner
2023PA-0000000224	Molecular Basis for Maternal Inheritance of Human Mitochondrial DNA	Dmitry	Temiakov
2023PA-0000000226	Determining the In Vivo Impact of Mitochondrial Energy Loss in the Liver	Cameron	Menezes
2023PA-0000000230	Temporary Treatment of Leigh Syndrome Reveals Clues to its Pathogenesis	Ernst-Bernhard	Kayser
2023PA-0000000236	The Role of C. elegans Metaxins in Mitochondrial Homeostasis	Jonathan	Dietz
2023PA-0000000237	Mitochondrial Ribosome Signaling and Survival Mechanisms in Mitochondrial Disease	Conor	Ronayne
2023PA-0000000238	Implementing a Novel No-Cost Genetic Testing Program for Patients with a High Likelihood of Having Primary Mitochondrial Disease	Philip	Yeske
2023PA-0000000239	mitoSHARE: a World-wide Patient-populated Registry for Mitochondrial Disease Patients and their Caregivers	Philip	Yeske

Abstract #: 2023PA-0000000113

Presenter: Jirair K. Bedoyan

Progress Towards Novel Target-Based Small Molecule Therapeutics for Pyruvate Dehydrogenase Complex Deficiency due to Specific Recurrent E1α Amino Acid Replacements

Jirair K. Bedoyan^1,2, Hatice Gokcan³, Polina Avdiunina, P.³, Robert James Hannan¹, Olexandr Isayev³

¹Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15219, USA, ²Division of Genetic and Genomic Medicine, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, 15224, USA, ³Department of Chemistry, Mellon Institute, Carnegie Mellon University, Pittsburgh, PA, 15213, USA

Introduction: The mammalian mitochondrial matrix multienzyme pyruvate dehydrogenase complex (PDC) is the gateway for oxidative metabolism of carbohydrates, catalyzing oxidative decarboxylation of pyruvate into acetyl-CoA as the primary substrate for the tricarboxylic acid cycle and oxidative phosphorylation. PDC is related structurally and functionally to other NAD-dependent multienzyme dehydrogenase complexes such as the α-ketoglutarate dehydrogenase and branched-chain α-ketoacid dehydrogenase (BCKDH) complexes, where functional (enzymatic) deficiencies result in human disease. Pyruvate dehydrogenase complex deficiency (PDCD) is a major cause of primary lactic acidosis resulting in high morbidity and mortality, with variable clinical presentation and an annual incidence in USA of at least 1 in 40,000 live births implying that at least 90 newborns annually will have PDCD. Therapeutic options for PDCD are limited. Ketogenic diet (KD) is currently the main untargeted intervention for primary-specific PDCD due to PDHA1 with benefits noted in areas of epilepsy, ataxia, speech/language development, and frequency of hospitalizations, but KD still leaves patients with significant systemic disease and developmental disability. Consequently, there is significant unmet needs for novel therapeutics for PDCD. Gene product-specific target-based small molecule therapeutics has not been explored for PDCD. Mutations in PDHA1 are responsible for ~85% of PDCD cases, with 55-60% due to disease-causing missense mutations (DMMs) and arginine (R) residue replacements constituting ~40% of all cases with DMMs. The E1 component of the multienzyme PDC (PDC-E1) is a symmetric dimer of heterodimers (αβ/α’β’) encoded by PDHA1 and PDHB, with two symmetric active sites each consisting of phosphorylation-Loops A and B.

Methods and Results: We measured solvent accessibility surface area (SASA), utilized nearest-neighbor analysis, and performed molecular modeling and molecular dynamic (MD) simulations including backbone mobility, principal component (PC), cluster, and eigenvector centrality analyses of wildtype (WT) PDC-E1 and PDC-E1 variants with E1α DMMs to investigate implications on PDC-E1 structure and function, and pathogenesis mechanism of specific E1α DMMs. SASA showed that >84% of residues with known non-duplicate DMMs of either E1α or E1β are solvent inaccessible (“buried”) and 30% of buried E1α DMMs are deleterious through perturbation of subunit-subunit interface contact (SSIC), with SSIC disrupting E1α R349 replacements (either R349H or R349C) constituting ~10% of all cases with DMMs. We find that mobility of phosphorylation-Loop A changes with certain DMMs including the E1α R349 replacements. PC analysis showed that essential motions for PDC-E1 biological activity changed with DMMs and that DMMs disrupted the communication network of WT PDC-E1, indicating allosteric effect in PDC-E1 structure and function. We identified a well-defined “E1α R349 pocket” in PDC-E1 variants with E1α R349H or R349C replacement as target for small molecule binding that could alter function of mutant PDC. Consequently, we have 1) virtually screened ~190,000 CNS-focused small molecules by machine learning trained on experimentally verified blood-brain barrier (BBB)+/- small molecules, 2) target “pocket” docked ~6,100 screened compounds including FDA-approved small molecules, 3) selected ~610 compounds with the highest docking scores for further analysis, and 4) identified BBB-permeable small molecules that theoretically bind in the “E1α R349 pocket” with ΔΔG <-8.5 kcal/mol (i.e., K_d <1 µM). We are evaluating these lead compounds for their ability to restore partially or fully, theoretical mutant PDC-E1 dynamics to WT equivalent dynamics by MD simulations and/or experimental in vitro activity of mutant PDC to WT equivalent levels using cultured fibroblasts from patients with PDCD due to E1α R349 replacements.

Conclusions: This work is the first proof-of-concept approach for novel E1α-specific target-based small molecule therapeutics for this highly disabling disorder and could pave the way for similar novel therapeutics approaches for other NAD-dependent dehydrogenase complex deficiencies important in human disease such as the BCKDH deficiency which results in maple syrup urine disease.

Abstract #: 2023PA-0000000114

Presenter: Gabriel C. Lander

DELE1 oligomers revealed by cryo-EM structure promote the integrated stress response activation

Jie Yang¹, Kelsey Baron², Daniel Pride¹, Anette Schneemann¹, Wenqian Chen¹, Xiaoyan Guo³, Martin Kampmann³, R. Luke Wiseman², Gabriel C. Lander^1,*

¹Department of Integrative, Structural and Computational Biology, Scripps Research, USA; ²Department of Molecular Medicine, Scripps Research, USA; ³Institute for Neurodegenerative Diseases, University of California, San Francisco, USA

^*Correspondence to: Gabriel C. Lander glander@scripps.edu

Mitochondria are dynamic organelles that must continually adapt and respond to cellular stress, and perturbations in mitochondrial function trigger the integrated stress response (ISR). Recent studies demonstrated that mitochondrial stress is relayed from the mitochondrial interior to the cytosol by the release of a C-terminal proteolytic fragment of DELE1 that binds to the HRI kinase to initiate integrate stress response signaling. Here, we report the cryo-electron microscopy structure of the active, C-terminal cleavage product of human DELE1 at ~3.8 Å resolution, revealing that DELE1 assembles into a high-order oligomer. We confirmed that active DELE1 similarly assembles into large oligomers in cells. This oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of distinct hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. Further, we show that assembly-impaired DELE1 mutants decrease the potential for this protein to induce ISR activation in cell culture models. Together, these findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR signaling following mitochondrial insult.

Abstract #: 2023PA-0000000116

Presenter: Heather Pomella

Outpatient Arginine Infusions as a Preventive Treatment for Stroke-Like Episodes in MELAS Syndrome: Early Results and Family Experience

Heather Pomella* MSN, RN, CPNP-PC, CNRN, Irina Anselm* MD, Lance Rodan* MD.

Heather.pomella@childrens.harvard.edu, Irina.anselm@childrens.harvard.edu, Lance.rodan@childrens.harvard.edu

Boston Children’s Hospital, Department of Neurology

MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome is a maternally inherited mitochondrial disease with a broad spectrum of manifestations. One of the most devastating complications of MELAS is recurrent stroke-like episodes. The pathophysiology of stroke-like episodes has not been entirely elucidated but is believed to involve a combination of energy failure from mitochondrial dysfunction and altered cerebrovascular perfusion. Individuals with MELAS syndrome have reduced plasma arginine levels, and levels further decrease during stroke-like episodes. Arginine is a nitric oxide precursor, and its low levels may relate to the abnormal cerebrovascular perfusion seen in this disorder. The use of oral L-arginine as maintenance therapy to reduce frequency of stroke-like episodes, and intravenous arginine as acute treatment for stroke-like episodes are now utilized as treatment in MELAS syndrome. During the acute stroke-like episode, patients receive a bolus of intravenous Arginine (500 mg/kg for children or 10 g/m² body surface area for adults) followed by the administration of similar dosage of intravenous Arginine for the next 3 to 5 days (Almannai, Hattab, Scaglia 2017). Oral L-arginine (150-300 mg/kg/day) is used to maintain plasma Arginine levels >168 umol/L chronically.

We care for a now 26-year-old woman with MELAS due to m.3271 A > C mutation with 60% heteroplasmy on her blood. She had been on oral L-arginine therapy since her diagnosis at the age of 13 years. From her diagnosis to the age of 24 years, she had 77 emergency room visits for concerns regarding metabolic stroke. Most times stroke-like lesions were confirmed on brain MRI, and she was admitted to hospital for IV arginine infusions. Over time, she progressively regressed in her cognitive abilities and developed moderate intellectual disability. Despite treatment with a higher than standard dose of oral L-arginine (500 mg/kg) combined with oral L-citrulline (100mg/kg), her plasma arginine levels remained below target range of 168 umol/L

At the age of 25 years we initiated outpatient biweekly intravenous arginine infusions in addition to continuing oral L-arginine therapy. Since initiation of outpatient arginine infusions in the past 12 months, our patient’s plasma arginine levels have been more consistently maintained above 168 umol/L. She has been tolerating the arginine infusions well, although has required additional bicarbonate supplementation to maintain normal bicarbonate levels (protocol of Arginine infusions, additional supplementation with bicarb will be provided). Since starting this regimen, she has only had one ER visit. Furthermore, caregivers report subjective improvement in her overall level of functioning and cognition. Her brain MRI to check on results of above therapy will be available soon and added to presentation along with previous MRIs.

We suggest that supplementation with IV arginine infusions in combination with oral L-arginine may be considered in select MELAS patients in whom it is difficult to maintain therapeutic plasma arginine levels and who continue to have frequent stroke-like episodes.

Abstract #: 2023PA-0000000117

Presenter: JW Adams

PPARδ Agonist MAvodelpar (REN001) Improves Mitochondrial Function In Skeletal Muscle: A Potential Treatment For Primary Mitochondrial Myopathies

Adams JW^1*, McCarrick M², and O’Carroll C^1,3

¹Reneo Pharmaceuticals, Inc., Irvine, California, USA; ²Plexium, Inc., San Diego, California, USA; ³Acadia Pharmaceuticals, Inc., San Diego, California, USA

*jadams@reneopharma.com

Background: Peroxisome proliferator–activated receptor delta (PPARδ) is a nuclear hormone receptor that transactivates genes required for mitochondrial respiration and oxidative metabolism. In skeletal muscle, PPARδ agonists enhance fatty acid oxidation (FAO) and mitochondrial biogenesis by upregulating the expression of genes involved in mitochondrial function and increasing muscle endurance. Primary mitochondrial myopathies (PMM), characterized by skeletal muscle weakness and fatigue due to dysfunctional mitochondria, cause impaired mobility and exercise intolerance. Therefore, PPARδ agonists may be attractive therapeutic candidates for the treatment of PMM. The objective of the analyses described here is to establish the potential utility of PPARδ agonist mavodelpar (REN001) for PMM.

Methods: Computational modeling, in vitro, and in vivo studies were used to establish the potency and selectivity of mavodelpar activation of PPARδ and transactivation of genes required for mitochondrial function in skeletal muscle.

Results: Computational modeling demonstrates that mavodelpar fits selectively into the binding pocket of PPARδ. Nuclear transactivation assays demonstrate mavodelpar is a potent and selective agonist of human and cynomolgus monkey PPARδ in recombinant cell lines (EC50 = 31 nM and 6.6 nM, respectively). Mavodelpar increases FAO in rat and human skeletal muscle cell lines in a dose-dependent manner without any observed effect on glycolysis. After oral dosing of mavodelpar in mice, mean (± SEM) skeletal muscle mRNA expression of carnitine palmitoyltransferase 1B (CPT1B), PPAR gamma coactivating factor 1 alpha (PGC-1α), pyruvate dehydrogenase kinase 4 (PDK4), and mitochondrial uncoupling protein 3 (UCP3) were 1.35 ± 0.15, 1.65 ± 0.19, 1.88 ± 0.17, and 2.29 ± 0.27-fold over vehicle, respectively. In rats, mean (± SD) skeletal muscle mRNA expression of angiopoietin-like 4 (ANGPTL4) increased by 5.5 ± 2.7- fold over vehicle (p < 0.01).

Conclusions: Mavodelpar is a potent and selective PPARδ agonist that increases expression of PPARδ-regulated genes for fatty acid metabolism, oxidative phosphorylation, and mitochondrial biogenesis in skeletal muscle. This pharmacological profile supports the evaluation of mavodelpar as a potential treatment for PMM.

Abstract #: 2023PA-0000000118

Presenter: Grainne Gorman

Mavodelpar Clinical Development Program In Adult Patients With Primary Mitochondrial Myopathy (Pmm): Results From Phase 1b Study And Design Of Ongoing Pivotal Study (Stride)

Pitceathly RDS^1,2, Stefanetti RJ^3,4, Blain A^3,4, Alcock L⁵, Newman J^3,4, Layton G⁶, Regan N⁷, Purkins L⁷, Davies M⁷, Dorenbaum A⁸, Mancuso M⁹, Karaa A¹⁰ and Gorman GS^3,4*

¹Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK; ²NHS Highly Specialised Service for Rare Mitochondrial Disorders, Queen Square Centre for Neuromuscular Diseases, The National Hospital for Neurology and Neurosurgery, London, UK; ³Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK; ⁴NIHR Newcastle Biomedical Research Centre, Newcastle University, Newcastle upon Tyne, UK; ⁵Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK; ⁶Paramstat Ltd., UK; ⁷Reneo Pharma Ltd., UK; ⁸Reneo Pharmaceuticals Inc., USA; ⁹Department of Clinical and Experimental Medicine, Neurological Institute, University of Pisa, Italy; ¹⁰Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

*Presenting author; Corresponding author’s email: Madhu Davies, mdavies@reneopharma.com

Primary mitochondrial myopathies (PMM) are rare, genetic disorders with defects of oxidative phosphorylation affecting predominantly skeletal muscle. We evaluated mavodelpar (REN001), a selective peroxisome proliferator-activated receptor delta (PPARδ) agonist, in an open-label Phase 1b study and we are currently conducting STRIDE (NCT04535609), an ongoing randomized (1:1), double-blind, placebo-controlled, pivotal 24-week trial to evaluate efficacy and safety of mavodelpar (100 mg PO QD) in adults with PMM with confirmed mtDNA defects (N~200). The open-label Phase 1b study enrolled 23 patients with genetically confirmed PMM to evaluate safety, tolerability, and exploratory efficacy endpoints of mavodelpar 100 mg administered once-daily orally for 12 weeks. Of the 23 participants, 17 completed 12 weeks of treatment (5 withdrew due to the Covid-19 pandemic, 1 withdrew consent). Mean (range) age was 54.5 (43–69) years; 65% were female; 48% had the m.3243A>G mutation. Most frequently reported treatment emergent adverse events (TEAEs) (n=4 for each) were constipation and headache. One treatment emergent serious adverse event (TESAE) of hematoma post muscle biopsy was reported. No deaths were reported. For completers at Week 12, baseline mean (range) distance walked in the 12-minute walk test (12MWT) of 603.2 m (range: 154 to 1013 m) improved by +104.4 m (95% CI: 53.1, 155.6); baseline mean weight-adjusted peak oxygen consumption of 14.594 mL/kg/min improved by +1.668 mL/kg/min (95% CI: -0.329, 3.665); baseline mean sit-to-stands (30STS) of 6.9 increased by +1.6 (95% CI: 0.0, 3.3); baseline mean SF-36 energy/fatigue score of 27.9 increased by +11.2 points (95% CI: 1.1, 21.2); baseline mean modified fatigue impact scale (MFIS) total score of 50.4 decreased (improved) by -10.5 points (95% CI: -16.3, -4.6); Brief Pain Inventory (short form) baseline mean severity and interference scores of 3.72 and 3.18 decreased (improved) by -0.81 (95% CI: -1.56, -0.06) and -0.26 (95% CI: -1.10, 0.58), respectively. The Phase 1b study results supported the ongoing development of mavodelpar in PMM, and informed the design of REN001-201 (STRIDE). Target enrollment in STRIDE was achieved in March 2023, having recruited subjects from sites in US, Canada, Europe, Australia, and New Zealand. The primary endpoint is change from baseline at Week 24 in the distance walked in the 12MWT. Secondary/exploratory endpoints include MFIS Physical score, Patient Global Impression of Change muscle symptom score, Patient Reported Outcomes Measurement Informal System (PROMIS), and other patient reported outcomes. Top-line results from the pivotal STRIDE study regarding the potential of mavodelpar to treat PMM, a disease with high unmet need and no approved treatment options, are anticipated Q4 2023.

Abstract #: 2023PA-0000000119

Presenter: Margaret Moore

Importance Of Patient And Caregiver Voice For Living With Primary Mitochondrial Myopathies And Associated Multi-Organ Involvement

Yeske P¹, Genova N², Simeone D², Moore M^1*, Martini D³, Babinski S⁴, Verma N⁴, Sapiro N⁴, Parikh S⁵

¹United Mitochondrial Disease Foundation, Pittsburgh, PA, USA; ²Astellas Pharma US LLC., Northbrook, IL, USA; ³Astellas Pharma Europe Ltd., Surrey, UK; ⁴Throughline Strategy Inc., Toronto, Canada; ⁵Mitochondrial Medicine Center, Neuroscience Institute, Cleveland Clinic, Cleveland, OH, USA

*Margaret.Moore@umdf.org

There are limited data available about the experience of primary mitochondrial myopathies (PMM) and its associated conditions for patients and their caregivers. This study explored patients’ and caregivers’ perspectives from symptom onset to PMM diagnosis and their experiences of navigating life with PMM. Four adults (30–65 years, with a diagnosis of CPEO + MM; CPEO + KSS; MELAS; and MERRF, respectively) and two caregivers (patients aged 25–30 years with MERRF and MELAS, respectively) were identified via UMDF in February 2022 to participate in individual semi-structured interviews. Questions were structured to be broad and open-ended to allow patients to discuss the key points they considered important. Participants were asked to describe their experiences living with the condition, including symptoms and disease-related impact on well-being, and with healthcare services before and after diagnosis. Survey participants reported a wide variability in symptoms from visual, to muscular, to stroke-like episodes/neuropathy. Some symptoms were common to nearly all, including exercise limitations, weakness/pain, vision problems, slurred speech/cognitive difficulty, and fatigue. The visibility of symptoms compounded the burden, causing patients to feel vulnerable to discrimination and social exclusion due to a lack of recognition of the disease by peers, family, friends, and employers. The unpredictable nature of symptoms, which changed daily, made it hard for patients and caregivers to plan and participate actively in daily life. This research found that patients and caregivers appear to lack clear mental models about how mitochondrial diseases work and what is going on in their bodies. Patients reported that they struggled to describe their condition accurately to healthcare teams, a finding compounded by the general lack of awareness of PMM in the medical community and consistent with a previous report.¹ To explain their condition, patients with PMM may refer more to their comorbidities, for which they have clearer mental models. Patients reported that confirmed genetic diagnoses could take years and were often the result of a fortunate convergence of self-advocacy, independent research, and access to the right specialist. Once diagnosed with PMM, patients reported feeling validated, but ultimately helpless as PMM is not well known, and treatments are not available. Patients and caregivers had a long experience living with PMM: the mean time since diagnosis was approximately 8 years. The interviews found evidence that patients learned to fulfil their role as experts, actively pursuing information related to their disease and taking responsibility for coordinating their care. Although some patients could adapt their lifestyle to help manage the disease, the increased financial burden of managing their symptoms and their impact on their daily life in the absence of effective treatments was substantial. As PMM progresses, patients reported feeling more vulnerable and increasingly reliant on caregivers due to physical weaknesses experienced, leading to a loss of independence. In conclusion, this research highlights the profound impact of PMM among patients and caregivers.

Author disclosures

MM, SP, and PY have no disclosures.

NG, DM, and DS are employees of Astellas Pharma Inc.

SB and NV are employees of Throughline Strategy Inc.

NS is the founder and primary shareholder of Throughline Strategy Inc.

Funding

This study was initiated and funded by Astellas Pharma Inc., and conducted in collaboration with United Mitochondrial Disease Foundation by Throughline Strategy Inc. Medical writing support was provided by Glen Dorrington, PhD, of Lumanity Communications, and funded by Astellas Pharma Inc.

Abstract #: 2023PA-0000000120

Presenter: Allison Hanaford

Peripheral macrophages causally contribute to disease onset and progression in the Ndufs4(KO) model of Leigh syndrome

Hanaford AR¹, Khanna A², James K¹, Chen Y¹, Mulholland M¹, Kayser B¹, Truong V¹, Sedensky M^1,5, Morgan P^1,5, Kalia V^2,6, Baerchst N^1,6, Sarkar S^2,6, and Johnson SC^1,3,4,5,7,*

¹Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA; ²Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, WA, USA; ³Department of Laboratory Medicine and Pathology, University of Washington. Seattle, WA, USA; ⁴Department of Neurology, University of Washington, Seattle, WA, USA, ⁵Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA, ⁶Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA, ⁷Department of Applied Sciences, Translational Bioscience, Northumbria University, Newcastle, UK

*simon.c.johnson@northumbria.ac.uk

Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common paediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high dose pexidartinib, a CSF1R (Colony stimulating factor 1 receptor) inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(KO) mouse model of LS. CSF1R signalling is critical for the survival, proliferation, and function of macrophage populations, including microglia. Inhibition of CSF1R causes depletion of microglia and other macrophage populations, with lower doses considered to target primarily microglia and higher doses targeting circulating macrophage populations in addition to microglia. Response to pexidartinib was dose dependent, implicating the involvement of peripheral immune cells in addition to microglia in LS pathology. To elucidate the specific immune populations involved in LS pathogenesis, we used a targeted genetic tool, deletion of the CSF1R macrophage super-enhancer FIRE (Csf1rΔFIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1rΔFIRE allele ablates microglia in both control and Ndufs4(KO) animals, but onset of CNS lesions and sequalae in Ndufs4(KO) mice, including mortality, are only marginally impacted. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Histologic analysis of brainstem lesions reveals the presence of non-microglial macrophages—direct evidence of a causal role for peripheral macrophages in the CNS lesions characteristic of LS. These data demonstrate that elimination of microglia is not sufficient to prevent necrotic lesion formation and that peripheral macrophages play a key role in disease pathogenesis in Ndufs4(KO) mice. Clinical case reports and other data suggest immune modulatory therapies are efficacious in multiple mitochondrial diseases and that inflammation and the immune system may drive the pathogenesis of multiple mitochondrial diseases, indicating our findings may be relevant to mitochondrial diseases beyond LS. The data presented here have important implications for any future efforts to translate immune-targeting strategies into treatments for genetic mitochondrial disease.

Abstract #: 2023PA-0000000121

Presenter: M Roy

Quantification of Mitochondrial Heteroplasmy

Roy M¹*, Guo Y¹, Bromberg R¹, Bharadwaj A¹, Orlikowska M¹, Otwinowski Z¹, Borek D¹

¹Department of Biophysics, University of Texas Southwestern Medical Center, USA

*mahitha.roy@utsouthwestern.edu

The classic role of mitochondria is the generation of ATP by OXPHOS; heart cells consume the energy produced by the mitochondria of their cardiac muscle cells to contract muscles, allowing the heart to pump blood. Every cell in the human heart muscle contains thousands of mitochondria, each of which may contain multiple copies of the mitochondrial genome—a small, circular genome of 16,569 base pairs. Mitochondrial DNA (mtDNA) accumulates more mutations than its nuclear counterpart due to: 1) a higher error rate of the mitochondrial polymerase POLG than the DNA polymerases that replicate nuclear DNA; 2) limited DNA damage repair in mitochondria; and 3) different mechanisms of selection against mutations between mtDNA and nuclear DNA. This combination can lead to mitochondrial heteroplasmy, a phenomenon where a cell contains a mix of wild type mtDNA and variant mtDNA. Low levels of heteroplasmy are common, but the extent to which heteroplasmy affects cellular processes and tissue functions is currently unknown due to the scarcity of methods for quantitating heteroplasmy levels. We have developed a sensitive method, using an optimized sample preparation and an in-house computational pipeline, which can detect the number and type of heteroplasmic mutations. We tested this method on 16 human heart samples from donors of varying age, sex, and cardiovascular health, and determined the levels of heteroplasmy. We are currently investigating various correlates of heteroplasmy levels.

Abstract #: 2023PA-0000000122

Presenter: Hemangi Bhonsle

Neuron-specific CASK Loss in Mice Causes Epileptic Encephalopathy Associated with Impaired Cerebral Cortex Development and Mitochondrial Dysfunction

Bhonsle HS^1*, Mukherjee K¹, Goodkin HP², and Srivastava S^1,3

¹Fralin Biomedical Research Institute at Virginia Tech Carilion, Roanoke, VA, 24016; ²Department of Neurology, University of Virginia School of Medicine, Charlottesville, VA, 22908; ³Department of Internal Medicine, Virginia Tech Carilion School of Medicine, Roanoke, VA, 24016

hemangisbhonsle@vtc.vt.edu; Sarika_Srivastava@vtc.vt.edu

CASK (calcium/calmodulin-dependent serine protein kinase) is an X-linked intellectual disability gene in mammals that is ubiquitously expressed in all tissues. Constitutive CASK homozygous knockout mice were shown to exhibit perinatal lethality indicating that CASK is essential for survival in mammals. CASK heterozygous loss-of-function mutations in human females and mice produces microcephaly with pontine and cerebellar hypoplasia (MICPCH) phenotype, whereas CASK hemizygous loss-of-function mutations in human males causes the infantile-onset epileptic encephalopathy (EE), which is highly devastating and fatal. Thus, CASK mutation patients clinically exhibit a broad phenotypic spectrum including intellectual disability, autism spectrum disorder, MICPCH, EE, optic nerve hypoplasia, developmental delay, hypotonia, dystonia, sensorineural hearing loss, and gastrointestinal complications. The mechanisms underlying CASK associated pathologies remain unknown. The goal of our study is to better understand the molecular function of CASK and the etiopathogenic mechanisms of CASK-linked EE. Using the Cre-LoxP conditional knockout strategy, we generated a novel mouse model of epileptic encephalopathy by deleting CASK specifically from all neurons in the brain (CASK^NKO). We found that the CASK^NKO mice exhibit severe growth retardation and early infantile-onset recurrent tonic spasms and myoclonus beginning postnatal day 8 (P8) that progressively worsens with age, and the mice die before adulthood (i.e. P25). The overall size of CASK^NKO mice brain is ~35-40% small, along with a disproportionately small cerebellum and an isometrically small cortex compared to the age-and-sex-matched CASK^wildtype littermate control mice. At P18, the CASK^NKO mice exhibit decreased cerebral cortical thickness associated with an increased neuronal density as well as mis-expression of cortical layer-specific markers (i.e. CUX1 and TBR1) compared to the age-and-sex-matched littermate control mice suggesting an impaired cerebral cortex development. CASK^NKO mice also displayed abnormal EEG (electroencephalogram) activity, increased excitatory/inhibitory synapse ratio, and a marked increase in reactive astrogliosis in the cerebral cortex compared to the age-and-sex-matched littermate control mice suggesting that the CASK^NKO mice brain harbor structural and/or functional abnormalities as well as the astrocytes mediated response to acute neuronal injury. Additionally, the brain mitochondrial respiration was significantly reduced in the CASK^NKO mice compared to the age-and-sex-matched littermate control mice. Transmission electron microscopy analysis revealed a reduced number of mitochondria in the cerebral cortex of CASK^NKO mice compared to the control mice. Furthermore, the Western blot analyses revealed that steady-state levels of several nuclear encoded mitochondrial oxidative phosphorylation proteins as well as proteins involved in mitochondrial fission and fusion processes were reduced in the cerebral cortex of CASK^NKO mice compared to the age-and-sex-matched littermate control mice. Interestingly, an unbiased RNA sequencing analysis revealed that ‘mitochondrion’ is the topmost gene ontology term and molecular function altered in the brain of CASK^NKO mice compared to the age-and-sex-matched littermate control mice. Altogether, our findings suggest that neuronal CASK is essential for postnatal brain development in mice, and plays a critical role in regulating mitochondrial function and homeostasis.

Abstract #: 2023PA-0000000123

Presenter: Emily Warren

Inflammatory and Interferon Gene Expression Signatures in Patients with Mitochondrial Disease

Warren EB¹*, Gordon-Lipkin EM¹, Cheung F², Chen J², Mukherjee A², Apps R², Tsang JS^2,3, Jetmore J¹, Kruk S¹, Lei Y⁴, West AP⁴, McGuire PJ¹

¹Metabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; ²Center for Human Immunology, National Institutes of Health, Bethesda, MD, USA; ³Department of Immunobiology, School of Medicine, Yale University, New Haven, CT, USA; ⁴Department of Microbial Pathogenesis & Immunology, Texas A&M University, Bryan, TX, USA

*emily.warren@nih.gov (presenting author’s email)

Recent evidence suggests that mitochondrial dysfunction contributes to multiple inflammatory processes and inflammatory dysregulation. People with mitochondrial disease (MtD) are susceptible to metabolic decompensation and neurological symptom progression in response to infection, which may arise from a mitochondrially-driven elevation in inflammatory tone. We collected whole blood from a heterogeneous cohort of MtD patients (n=31, including pediatric and adult, male and female cases) and healthy controls (n=48) and performed RNAseq to examine transcriptomic differences. We used gene ontology and gene set enrichment analyses to identify commonly dysregulated pathways. Gene sets involved in inflammatory signaling, including type I interferons, interleukin-1β and antiviral responses, are enriched in MtD patients compared to controls. Monocyte and dendritic cell gene clusters are also enriched in MtD patients, while T cell and B cell gene sets are negatively enriched. The enrichment of antiviral response corresponds with similar immune response and inflammatory enrichments in an independent set of MELAS patients and two mouse models of mtDNA dysfunction. Further, we have identified a novel sex-specific pattern of immune gene expression changes, wherein males with MtD had negative enrichment of T cell and NK cell genes, while females had negative enrichment of B cell genes, and positive enrichment of interleukin-1β genes. Our dataset provides key clinical evidence linking MtD to inflammation, demonstrating translational support of the causative relationship between mitochondrial dysfunction and systemic peripheral inflammation. Dysregulation of these pathways may contribute to the pathogenesis of primary MtD and other chronic inflammatory disorders associated with mitochondrial dysfunction.

Abstract #: 2023PA-0000000124

Presenter: Kumarie Latchman

A Novel APOO Deletion in An African American Male With Epilepsy, Autism, Ataxia, Hypotonia and Cognitive Deficit

Latchman, K¹, Fontanesi, F²

¹Department of Clinical and Translational Genetics, University of Miami Miller School of Medicine, USA; ²Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, USA

*Kxl604@med.miami.edu

Mitochondrial disease due to mutation in genes responsible for the structural integrity and morphology of the mitochondrial inner membrane has been previously described. Mitochondrial cristae, formed from the invagination of mitochondrial inner membrane, hold the mitochondrial respiratory complexes and are the sites of oxidative phosphorylation. Cristae formation and maintenance is regulated by the MICOS (mitochondrial contact site and cristae organizing system) complex and defects in MICOS complex proteins have been previously reported in association with several mitochondrial diseases. The APOO (apolipoprotein O) gene encodes MIC26, one of seven protein subunits of MICOS, and a APOO missense mutation (I117T) was recently reported in association with X-linked recessive mitochondrial disease (mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features) with evidence of mitochondrial structural abnormalities, namely increased cristae width and decreased cristae density. The mechanism underlying this mitochondrial dysfunction phenotype remains to be elucidated. Others have shown that the absence of MIC26 in a yeast model was not rescued by the human mutant allele, APOO ( I117T), when compared to wild type. The primary goal of this research is to characterize mitochondrial dysfunction due to complete APOO deletion in a patient with high suspicion for a mitochondrial disease. Here, we present a six year-old African American male with epilepsy, autism spectrum disorder, ataxia, hypotonia and cognitive deficit and with a 275 kilobase multi-gene deletion on chromosome X, arr[GRCh37] Xp22.11 (23731277_24006696)x0. This maternally inherited deleted region, confirmed on clinical chromosomal microarray and next generation whole exome sequencing, includes APOO and four other genes, ACOT9, CXorf58, KLHL15 and SAT1, albeit with low suspicion for genetic etiology. Maternal history is significant for intellectual disability and psychiatric disorder, including bipolar disorder. Using patient-derived fibroblast obtained from skin punch biopsy, cells were cultured and immortalized. Both patient-derived and control fibroblasts were imaged by transmission electron microscopy. Leveraging knowledge gained from the previously reported mitochondrial dysfunction phenotype due to an APOO missense variant, we hypothesize that a complete APOO deletion will produce similar defects in the mitochondrial cristae. Our preliminary data shows grossly abnormal cristae morphology and a tendency to decreased cristae density and number. In summary, we have identified a novel APOO mutation associated with epilepsy and expanded the phenotypic and genotypic spectrum of APOO-related mitochondrial disease. Moreover, we have established a novel patient-derived cell line with a complete APOO deletion, which we anticipate will allow us to gain insights into the role of MIC26 in cristae morphology and organization.

Abstract #: 2023PA-0000000126

Presenter: Amanda Fuchs

Systemic cytokine storm exacerbates acute influenza A infection in Ndufs4 KO mouse

Fuchs AL^1*, Warren E¹, Franco JLM¹, Jetmore J¹, Thompson E^1,2, Schlein M¹, Kruk S¹, Gordon-Lipkin EM¹, Tarasenko TN¹, and McGuire PJ¹

¹ Metabolism, Infection, and Immunity Section, Metabolic Medicine Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America; ² Current address: Pathobiology & Mechanisms of Disease Program, Columbia University, New York, New York, United States of America

*amanda.fuchs@nih.gov

Influenza viruses are responsible for millions of contagious respiratory infections worldwide every year that in most cases only result in mild clinical symptoms. However, high risk populations, such as immunocompromised patients, are more likely to develop severe complications and increased mortality risk. Pediatric patients with mitochondrial disease (MtD) represent a medically vulnerable group with 89% of pediatric MtD patients suffering from recurrent upper respiratory infections that can trigger life-threatening, neurodegenerative events, known as metabolic decompensation. Therefore, it is imperative to investigate how MtD energetic deficits alter the immune response and influenza pathogenesis in a way that is ultimately detrimental to the host. Herein, we examined the impact of mouse-adapted human influenza A (H3N2) infection on Ndufs4 KO mice, a MtD mouse model of Leigh syndrome, using a whole-body inhalation exposure system. Our study revealed that Ndufs4 KO mice demonstrated worse clinical scores, reflected by elevated rates of body weight loss, excessive lung viral titers, and reduced splenic index during influenza infection. In addition, hematological analyses of peripheral blood from influenza infected Ndufs4 KO mice revealed erythrocytosis, polycythemia, and neutrophilia. Moreover, early systemic cytokine dysregulation was observed in Ndufs4 KO mice with increased levels of CCL2 (MCP-1), CCL5 (RANTES), CXCL1 (KC), CXCL10 (IP-10), GM-CSF, IFN-γ, IL-6, IL-12, and TNF-α consequent to influenza infection. Altogether, these results suggest that aberrant viral replication and early cytokine response dysregulation play a role in metabolic decompensation and disease exacerbation in MtD patients during seasonal influenza infection.

Abstract #: 2023PA-0000000128

Presenter: Neena Patel

A Case Study Of A Trio Of Siblings Utilizing Elamipretide For Polg1 Mitochondrial Disease

Patel N¹*, Miller L¹, Guerra W¹, Russo SN¹, and Koenig MK¹

¹University of Texas McGovern Medical School, Department of Pediatrics, Center for the Treatment of Pediatric Neurodegenerative Disease, Houston, Texas

*neena.patel@uth.tmc.edu

Elamipretide, an investigational product targeting the inner mitochondrial membrane, improves membrane integrity in dysfunctional mitochondria resulting in increased ATP production. In this case series, we reviewed the use of elamipretide in a trio of siblings diagnosed with phenotypic sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO) caused by compound heterozygous pathogenic variants in the POLG1 gene. POLG1 is integral to proper replication and repair of the mitochondrial DNA (mtDNA). As such, defective POLG1 function leads to an accumulation of deletions and depletion of mtDNA. Each of the three siblings with SANDO was introduced to elampretide at different ages and at different stages in their disease states. Each individual participated in a clinical trial of varying phases and study designs. Sibling 1, a 48-year-old male diagnosed with POLG1 mitochondrial disorder, began taking elamipretide through a phase 3, double-blind, placebo-controlled clinical trial (SPIMM-301) with a starting dose of 40 mg subcutaneously (SC) daily. Following the placebo-controlled phase, Sibling 1 transitioned to open-label therapy at 40 mg SC on January 24, 2018. While on this therapy, he reported improvement in his activities of daily living (ADLs), increased ambulation and decreased pain overall. Upon conclusion of the trial, therapy was withdrawn on January 8, 2020. The subject reported loss of all benefits within weeks. Sibling 1 resumed therapy on March 31, 2020 through an Expanded Access Program (EAP). He was initiated on 40 mg SC daily and on June 22, 2021 was titrated to a dose of 60 mg daily. Again, he gained function and noted decreased pain, but has since developed progressive decline. Overall, his clinical team believes this therapy has slowed the rate of his deterioration. Sibling 2, a 45-year-old female with POLG1 mitochondrial disorder, began taking elamipretide on March 29, 2022 through the EAP program at a dose of 40 mg daily. Sibling 2 was wheelchair bound at baseline due to her ataxia. She also reported short term memory loss and concentration concerns. Since beginning treatment, Sibling 2 noted an increase in her ability to self-transfer and get out of her wheelchair. She also reported improved memory and concentration. Sibling 3, a 40-year-old male, with POLG1 mitochondrial disorder, enrolled in a phase 3, double-blind, placebo-controlled clinical trial (SPIMD-301), examining the efficacy and tolerability of a 60 mg SC daily dose on April 29, 2022. He will complete this study in March 2023, at which time he too will transition into the EAP. Currently, the only adverse events reported by any of the siblings have been mild injection site reactions. This unique case of three siblings receiving elamipretide for their progressive mitochondrial disorder has allowed us to investigate the efficacy and tolerability of elamipretide on subjects with similar genetic profiles and only slight variability in clinical presentation. All have demonstrated improvements in their condition with excellent tolerability of elamipretide.

Abstract #: 2023PA-0000000129

Presenter: Joanna Rorbach

Loss of pathogenic mitochondrial tRNA mutations during the development of adaptive immune responses

Jingdian Zhang^1,2, Camilla Koolmeister^1,2, Jinming Han³, Roberta Filograna^1,2, Martin Engvall⁴, Anna Wredenberg^1,2,4, Gunilla B. Karlsson Hedestam⁵, Xaquin Castro Dopico⁵, Joanna Rorbach^1,2*

¹Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 17165, Sweden; ²Max Planck Institute Biology of Ageing-Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm 17165, Sweden; ³Applied Immunology and Immunotherapy, Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska University Hospital, Stockholm 17176, Sweden; ⁴Center for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm 17164, Sweden; ⁵Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm 17177, Sweden

*Corresponding author email: Joanna.rorbach@ki.se

Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) can exhibit heteroplasmy and cause a range of multi-syndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect hematopoietic cell function is largely unknown. Here, in mice carrying heteroplasmic, pathogenic C5024T in mt-tRNA^Ala, and Mitochondrial Encephalomyopathy, Lactic Acidosis, Stroke-like episodes (MELAS) syndrome patients, carrying pathogenic A3243G in mt-tRNA^Leu, we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naïve counterparts. Pathogenic burden reduction was less pronounced in myeloid compared to lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor-triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8⁺ T cell mitochondrial respiration, metabolic remodeling, and IFN-γ production post-activation. Together, our data illustrate that the generation of immunological memory shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

Abstract #: 2023PA-0000000130

Presenter: Natalie Niemi

The mitochondrial phosphatase Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy

Niemi N.M.^1*

¹ Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, USA

*niemi@wustl.edu (*Corresponding author’s email)

Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within this organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 exhibit severe metabolic deficiencies, including hypoglycemia, hypoketosis, and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7^-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Follow up studies have linked this decrease in mitochondrial protein content to excessive mitophagy via the stabilization of two mitophagy receptors, Bnip3 and Nix. Consistently, Pptc7^-/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs—including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways, and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

Abstract #: 2023PA-0000000131

Presenter: Yentli Soto Albrecht

Non-pathogenic variation in mitochondrial DNA modulates murine SARS-CoV-2 pathogenesis

Soto Albrecht Y.E.^1-4*, Kenney D.^3,4, Morrow R.², Olali A.Z.², Tseng A.^3,4, Lo M.^4,5, O’Connell A.^4,5, Huang J.², Mitchell K.L.², Gertje H.P.^4,5, Sheikh A.^3,4, Yardeni T.², Murdock D.G.², Angelin A.², Hancock W.⁶, Belkina A.C.^5,7, Crossland N.A.^4,5, Douam F.^3,4, Wallace D.C.^2,8.

¹Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; ²Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA; ³Department of Microbiology, Boston University School of Medicine, Boston, MA; ⁴National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA; ⁵Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA; ⁶Department of Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; ⁷Flow Cytometry Core Facility, Boston University School of Medicine, Boston, MA; ⁸Division of Human Genetics, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

*Yentli.SotoAlbrecht@pennmedicine.upenn.edu

Genetic determinants of COVID-19 outcomes are of increasing interest. Human genetic variation derives from nuclear and mitochondrial DNA (mtDNA). As humans radiated from Africa, beneficial functional mtDNA variants were selected and their descendant mtDNAs formed ‘haplogroups’. mtDNA haplogroup has recently been linked to the age of onset of critical COVID-19. However, a causative link between host mtDNA background and SARS-CoV-2 pathogenesis has not been established. To model the breadth of human mtDNA variation, our lab generated mice containing murine mitochondrial haplogroups 129 or NZB mtDNA on the K18-hACE2 nuclear background of severe SARS-CoV-2 pathogenesis. Importantly, 129 and NZB mtDNA differ from the wild-type (WT) C57BL/6 reference by 2 and 91 base-pairs respectively. We found that K18-hACE2 mice harboring 129 or NZB mtDNA (hereby 129 and NZB) both showed increased survival (40%) over WT mice (13%) by 14 days post infection (DPI) when challenged intranasally with SARS-CoV-2. NZB mice additionally demonstrated delayed onset of clinical disease, where protection correlated with increased splenic B cells and monocyte, macrophage, and CD8 T cell lung infiltrates, paired with decreased splenic, lung and peripheral T regulatory (T reg) cells, at 7DPI. NZB T reg cells showed impaired suppressive function in separate in vivo studies. However, there was no difference in lung viral replication across genotypes. The mtDNA contribution to disease was explored in a secondary mouse-adapted virus model of SARS-CoV-2 pathogenesis (MA30). Improved clinical disease in NZB mice was also observed, where protection correlated with decreased lung viral replication at 2DPI. We show that mitochondrial haplogroup alone can influence disease in two murine models of severe SARS-CoV-2 infection, where multiple paths to improved clinical disease exist, including pro-inflammatory immunophenotypes and restricted viral replication. Our discovery contributes to improved understanding of how common variation in host factors can impact the severity of COVID-19.

Abstract #: 2023PA-0000000133

Presenter: Nicole Defoor

Remdesivir Increases Mtdna Copy Number Causing Mild Alterations To Oxidative Phosphorylation

DeFoor N¹, Paul, S², Li S³, Basso E K G⁴, Stevenson V², Browning J L¹, Prater A K¹, Tao G³, and Pickrell A M^1*

¹School of Neuroscience, Virginia Tech, Blacksburg, VA, USA, 24061; ²Graduate Program in Biomedical and Veterinary Sciences, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA, 24061; ³Department of Regenerative Medicine & Cell Biology, Medical University of South Carolina, Charleston, SC, USA, 29425; ⁴Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA, 24061

*alicia.pickrell@vt.edu

SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it has been metabolized in the liver. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA) itself. For instance, the human immunodeficiency virus (HIV) treatment drug Zidovudine (AZT) works as a thymidine analogue to halt viral DNA transcription during reverse transcription. However, patients treated with AZT years later displayed myopathies, which was later discovered to be due to off-target mitochondrial damage from both short and long-term usage. Similarly, Fialuridine (FIAU) was tested for the treatment against hepatitis B virus (HBV) but did not pass Phase II clinical trials due to unforeseen mitochondrial damage. These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated the study to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu lung cells and rodent liver 30 days post-treatment. However, these increases only resulted in mild changes to mitochondrial function. Nuclear-encoded oxidative phosphorylation (OXPHOS) complex II protein expression was decreased in Mv1Lu cells, yet proteins for other OXPHOS complexes remained unchanged in Mv1Lu cells and rodent liver. However, mitochondrial transcription factor A (TFAM) protein expression was decreased in RDV treated liver tissue. Complex IV activity was unaffected, while citrate synthase activity was significantly increased in our RDV treated liver. We tested liver toxicity of RDV by measuring levels of AST and ALT enzymes in the blood serum. Though heightened levels of these enzymes indicate damage to the liver, we found no changes with RDV treatment. This lack of toxicity aligned with the pathology of liver tissue, in which we found no lesions. Next-Generation Sequencing found no substantial changes in the mtDNA mutational load. Surprisingly, skeletal muscle and heart were extremely resistant to RDV 30 days post-treatment, tissues that have previously been affected by antivirals. mtDNA copy number and mitochondrial protein expression were unchanged. Cardiac function and histology were unremarkable. Induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes also displayed a strong resistance to RDV treatment. Although our data suggests that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.

Abstract #: 2023PA-0000000134

Presenter: Stephen Ekker

Harnessing the potential of transcriptional adaptation as a mechanism for mitochondrial genetic disorders

Morales Gomez A¹, Staff N², Ekker SC³

¹Mayo Clinic Graduate School of Biomedical Science, Mayo Clinic, USA; ²Department of Neurology, Mayo Clinic, USA; ³Department of Biochemistry and Molecular Biology, Mayo Clinic, USA

*ekker.stephen@mayo.edu

CHCHD10 is a nuclear gene that encodes a mitochondrial protein that enhances cristae junctions. Genetic variation in CHCHD10 can lead to rare mitochondrial dysfunction in amyotrophic lateral sclerosis (ALS), but the mechanism underlying this differential clinical manifestation due to genetic variation is unknown. One recent genetic compensatory mechanism called transcriptional adaptation (TA) is becoming recognized as a major mechanism underlying differential phenotypic genetic responses in animal models. TA functions through a distinct process whereby an abnormal premature termination codon undergoes nonsense-mediated mRNA decay followed by the identification of promoter regions in homologous gene(s) that get upregulated. Our study focuses on testing whether CHCHD10 variant and CHCHD2 upregulation compensation is due to TA and whether this approach operates through mitochondrial function. To examine this, we utilized gene editing tools in HT1080 cells and patient samples with known CHCHD10 mutations causative for ALS. Our approach would advance discovery science towards by exploring CHCHD10/2 TA mechanism that can lead to novel therapies for rare mitochondrial disorders such as CHCHD10-mediated neurodegenerative disease.

Abstract #: 2023PA-0000000135

Presenter: NC Kim

FDA-approved PDE4 inhibitors reduce the dominant toxicity of ALS–FTD-associated CHCHD10^S59L in Drosophila and human cells

Baek M¹†, Choe YJ¹†, and Kim NC¹*

¹Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, USA

*kimn@umn.edu

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) are a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS-FTD). Using in vivo Drosophila models expressing C2C10H^S81L, and human cell models expressing CHCHD10^S59L, we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10^S59L. Therefore, we have further evaluated various additional small molecule compounds that can modulate the PINK1/Parkin pathway and reduce CHCHD10^S59L-induced cytotoxicity. Among these compounds, FDA-approved PDE4 inhibitors successfully reduced CHCHD10^S59L-induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10H^S81L. Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10^S59L via the cAMP-PKA pathway. These findings suggest that PDE4 inhibitors currently available in the market can be repositioned to treat CHCHD10^S59L-mediated ALS-FTD and possibly other related diseases.

Abstract #: 2023PA-0000000136

Presenter: L. Miller

Emergency Use of doxectine and doxribtiminein an Adult Patient with TK2d Following a Traumatic Fall

Miller L¹*; Clearman A¹, Hill C¹, Koenig M K¹; Russo S N¹

¹University of Texas McGovern Medical School, Department of Pediatrics, Center for the Treatment of Pediatric Neurodegenerative Disease, Houston, Texas

*lindsey.b.miller@uth.tmc.edu

We report the case of a 26-year-old female with thymidine kinase 2 (TK2) deficiency due to compound heterozygous pathogenic variants, c.361C>A and c.547C>T, in the TK2 gene. Initial symptoms of muscle pain and fatigue began at age 10 years of age. The patient experienced progressive functional decline with acute decompensation after a respiratory infection at 24 years of age. Following this illness, she became wheelchair bound requiring intermittent BiPAP support. She also developed a decrease in appetite with a concurrent 20-pound weight loss. She developed COVID-19 in March 2021 resulting in increased use of BiPAP. In August of 2021 (25 years of age), the patient sustained bilateral sacral fractures after a fall in her home. Respiratory distress worsened and she was started on nucleoside therapy with a combination of doxecitine and doxribtimine, under expanded access due to impending respiratory failure. Doxecitine and doxribtiminewas started at 260 mg/kg/day and up-titrated to a target of 800mg/kg/day. Weight is checked at clinic visits every 3 months and the daily dose is adjusted accordingly. An adverse event of diarrhea was observed with dosage increases, now resolved. Since initiating doxecitine and doxribtimine, the patient has resumed the ability to perform most of her activities of daily living (ADLs) independently. BiPAP use has been weaned to night time use only. After 18 months the patient reports improvement in overall health on Quality of Life (QOL) self-assessments, ranking her overall health at 82/100. Revised Upper Limb Module score has improved from 19 to 36. She is currently able to ambulate 25 feet with a walker with stand by assistance before needing to rest. The patient reports that she has now returned to activities that she was not able to do even before her most recent fall such as driving and going to social events. Doxecitine and doxribtimine improved our patient’s quality of life and decreased her medical dependence as evidenced by improved ability to perform her own activities of daily living and decreased need for day- time respiratory support. Doxecitine and doxribtimine is an investigational drug that has not been approved by FDA or any other regulatory agency. This is a non-controlled and observational report with an investigational drug. Special thank you to UBC for providing investigational product doxecitine and doxribtimine under IND # 157831 and supporting the EAP under IRB # HSC-MS-21-0675 at The University of Texas McGovern Medical School, Center for the Treatment of Pediatric Neurodegenerative Disease.

Abstract #: 2023PA-0000000137

Presenter: RJ Snyder

Hemin Impairs Mitochondrial Gene Expression and Induces Guanine Quadruplexes in Human Renal Proximal Tubule Cells

Snyder RJ^1* and Watts JA¹

¹ Epigenetics and Stem Cell Biology Laboratory, NIEHS, NIH

*snyder3@niehs.nih.gov (*Corresponding author’s email)

Fidelity of mitochondrial gene expression is essential for oxidative phosphorylation and cellular function. Dysregulation of mitochondrial gene transcription is known to contribute to the pathogenesis of kidney and other human diseases, yet our understanding of the regulation of RNA synthesis by mitochondrial RNA polymerase (mtRNAP) is incomplete. Previously, we observed that mtRNAP pauses frequently while transcribing mtDNA. These pauses are associated with guanine-rich sequences which form guanine quadruplex (G4) secondary structures. We and others have found G4 stabilization by the small molecule RHPS4 can impair transcription of mtDNA. In this study, we investigated the iron-binding porphyrin hemin as an endogenous G4-stabilizing agent in renal proximal tubule epithelial cells (RPTEC). Kidney epithelial tissue is exposed to high concentrations of hemin in pathologic states such as hemolysis or rhabdomyolysis. To determine whether hemin exposure affects G4 abundance, human primary RPTECs were exposed to increasing concentrations of hemin in serum-free culture. Immunofluorescence revealed a dose-dependent increase in intracellular G4 abundance after 1 hour with a pattern matching exposure to the known G4-stabilizer RHPS4. Expression of mitochondrial genes RNR2, MT-CO1, and MT-ND6 was significantly reduced while expression of nuclear-encoded mitochondrial transcription factors TFAM and TEFM was unaffected, indicating a selective effect on mitochondrial transcription. Hemin exposure did not induce mitophagy in RPTECs up to 20 µM (as measured by PINK1 expression and MtPhagy reporter dye) and did not significantly alter the total mitochondrial mass per cell (as measured by TOMM20 staining and SDHA expression). Nonetheless, mitochondrial membrane potential was impaired by hemin exposure and this effect persisted for at least 24 hours. These results support the hypothesis that hemin impairs transcription by mtRNAP through stabilization of guanine quadruplexes. We suggest that hemin-mediated impairment of transcription by mtRNAP may contribute to mitochondrial dysfunction in heme pigment nephropathy and other forms of acute kidney injury.

Abstract #: 2023PA-0000000138

Presenter: Joseph Guarnieri

Targeted Down Regulation Of Core Mitochondrial Genes During Sars-Cov-2 Infection

Joseph W. Guarnieri^1,2,3, Deborah G. Murdock^1,2, Alessia Angelin^1,2, Timothy Lie^1,2, Gabrielle A. Widjaja^1,2, Jeffrey Haltom^1,2,3, Shelly Robertson⁴, Stephen B. Baylin^3,8, Eve Syrkin Wurtele^3,6, Deanne Taylor^1,2,3, Sonja M. Best⁴, Christopher E. Mason^3,5,9, Jonathan C. Schisler^3,7, Robert E. Schwartz^3,5, Afshin Beheshti^3,10,11, Douglas C. Wallace^1,2,3,12

¹Center for Mitochondrial and Epigenomic Medicine, ²The Children’s Hospital of Philadelphia, Philadelphia, PA 19104 USA., ³COVID-19 International Research Team., ⁴Rocky Mountain Laboratory, National Institute of Allergy and Infectious Disease NIH, Hamilton, MT 59840., ⁵Weill Cornell Medicine, NY, 10065, USA., ⁶Iowa State University, Ames, IA 50011, USA., ⁷University of North Carolina, Chapel Hill, Chapel Hill, NC, 27599, USA., ⁸Johns Hopkins School of Medicine, Baltimore, MD 21287, USA., ⁹New York Genome Center, NY, USA., ¹⁰Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA., ¹¹KBR, NASA Ames Research Center, Moffett Field, CA, 94035, USA., ¹²Division of Human Genetics, Department of Pediatrics, University of Pennsylvania, Philadelphia, PA 19104 USA

WallaceD1@chop.edu (*Corresponding author’s email)

To investigate how SARS-CoV-2 affects oxidative phosphorylation (OXPHOS) and the innate immune response in vivo, we calculated the relative expression levels of host genes in RNA sequencing data from 700 nasopharyngeal samples and 40 autopsy cases from SARS-CoV2 positive and negative individuals, and infected hamsters and mouse models. Our data demonstrated that the virus inhibits OXPHOS nuclear DNA (nDNA) gene transcription by blocking coordinately expressed genes of modular components of the OXPHOS holoenzymes. While the initial nasopharyngeal infection is associated with the downregulation of OXPHOS genes, lung OXPHOS was upregulated at death. By contrast, heart, kidney, and liver nDNA gene expression remains suppressed, resulting in increased production of pro-inflammatory cytokines. This concerted inhibition of OXPHOS results in the activation of the innate immune response and activates HIF-1α inducing a pseudo-hypoxic state favoring viral biogenesis. These findings suggest that SARS-CoV-2 disrupts mitochondrial function to increase mitochondrial reactive oxygen species (mROS) production. Increased mROS production can activate the innate immune response and increase protein levels of HIF-1α. Increased HIF-1α triggers a shift in cellular metabolism to favor glycolysis redirecting carbon molecules from mitochondrial oxidation to viral assembly. Since suppressing mitochondrial function and increasing mROS are essential for viral pathogenesis. Interventions targeting mROS or improving OXPHOS may mitigate viral replication and COVID-19 severity.

Abstract #: 2023PA-0000000140

Presenter: Jean Flickinger

Validation Of The Mitochondrial Myopathy Function Scale

Flickinger J^1,2*, Santos JD², Ballance E^1,2, Martin I², Xiao R^3,4, Zolkipli-Cunningham Z^2,5

¹Department of Physical Therapy, Children’s Hospital of Philadelphia, United States of America; ²Mitochondrial Medicine Frontier Program, Department of Human Genetics, Children’s Hospital of Philadelphia, United States of America; ³Department of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania, United States of America; ⁴Department of Pediatrics, Division of Biostatistics, Children’s Hospital of Philadelphia, United States of America; ⁵Department of Pediatrics, University of Pennsylvania, USA

*flickingerj@chop.edu

Motor function scales to measure the impact of disease such as the North Star Ambulatory Assessment (NSAA) for Duchenne Muscular Dystrophy (DMD) patients have been essential for monitoring disease progression and treatment response. There are currently no existing Mitochondrial Myopathy (MM)-specific motor function measures. The MM-Composite Assessment Tool (MM-COAST) was the first objective outcome measure validated in MM that quantifies impairments in key domains of muscle strength, fatigue, exercise intolerance, dexterity, and balance. However, we are lacking a motor function scale that quantifies the functional consequences of having deficits in these key domains. We trialed the NSAA but observed ceiling effect as DMD patients are weaker than MM patients. This initiated our effort to develop the MM-Function Scale, to provide a global score of MM motor function. MM-Function Scale yields ordinal data on a scale of 0-3. After several iterations, we have validated the final MM-Function Scale in MM children and adults (n=58, mean age 26.6 years ± 17.9, 66 % female), including 44 (76%) with mtDNA and 14 (24%) nuclear genetic etiologies. Three of the 58 subjects were non-ambulatory. MM-Function Scale mean score (± SEM) for the MM cohort was 53.2 ± 2.44 (70.9%) of a possible total score of 75, a higher score indicating better overall motor function. There was no significant difference between child and adult scores (p=0.98), or between nuclear and mtDNA etiologies (p=0.94). We found a strong negative correlation between MM-Function Scale scores with our validated MM-COAST composite scores, where a high MM-COAST score indicates greater MM disease severity (r=-0.67, p<0.0001, n= 58). Specifically, MM-Function Scale scores significantly correlated with each MM-COAST domain z-scores of dominant elbow flexion strength (r=0.47, p=0.0004, n=52) and hip flexion strength (r=0.51, p=0.0001, n=51); repeated elbow flexion muscle fatigue assessment (r=0.42, p=0.003, n=47); balance testing single leg eyes closed (r=0.41, p=0.002, n=58) and tandem stance eyes open (r=0.68, p<0.0001, n=35); 9 hole peg test (r=0.68, p<0.0001, n=58) and functional dexterity test (r=0.49, p=0.0004, n=58); and exercise intolerance assessments of 30 seconds sit to stand (r=0.67, p<0.0001, n=45) and 6-minute walk test z-scores (r=0.67, p<0.0001, n=47). These significant correlations demonstrate that MM-Function Scale assessments are indeed representative of corresponding MM-COAST deficits. We have also designed a Mitochondrial Mobility Performance Level classification system to cluster patients by mobility status. MM-Function Scale mean score for subjects with Level 5 mobility (minimal fatigue: independent community level ambulator, walk one mile without rest, climb up 2 flights steps without rest) was 63.7 ± 10.6 (84.9%, n= 20) was significantly different compared to individuals with Level 4 mobility (moderate fatigue: independent community level ambulator, walk one mile but requires rest, climb one flight but not 2 flights without rest) scoring 51.1 ± 13.4 (68.2%, n=21), p= 0.002, demonstrating a greater functional impact in MM subjects with moderate fatigue. In conclusion, results indicate that our newly validated MM-Function Scale is clinically meaningful and has capability to quantify MM motor function and may hold particular utility in characterizing disease trajectory and treatment response in future studies.

Abstract #: 2023PA-0000000143

Presenter: Jean Flickinger

Mitochondrial Mobility Performance Levels

Flickinger J^1,2*, Martin I², Santos JD², Xiao R^3,4, Zolkipli-Cunningham Z^2,5

¹Department of Physical Therapy, Children’s Hospital of Philadelphia, United States of America; ²Mitochondrial Medicine Frontier Program, Department of Human Genetics, Children’s Hospital of Philadelphia, United States of America; ³Department of Biostatistics, Epidemiology, and Informatics, University of Pennsylvania, United States of America; ⁴Department of Pediatrics, Division of Biostatistics, Children’s Hospital of Philadelphia, United States of America; ⁵Department of Pediatrics, University of Pennsylvania, USA

*flickingerj@chop.edu

Mitochondrial Myopathy (MM) presents with diverse symptoms of varying severity. Our initial attempts to characterize disease progression across an MM cohort using a validated outcome measure, the MM-COAST, indicate large variability in individual trajectories ranging from stability over time to significant decline or improvement, yielding a mean rate of change across the MM-COAST cohort that may be less informative. Subgroup analysis by age or genetic etiologies (nuclear vs. mitochondrial (mt)DNA) has not demonstrated significant differences. We developed the Mitochondrial Mobility Performance Levels (IMPROVE) system that assigns different levels of functional mobility dependent on prominence of muscle weakness, imbalance and fatigue. We hypothesized that clustering MM patients into subgroups sharing similar levels of mobility would enhance MM cohort data analyses and be clinically meaningful. The IMPROVE has 6 levels of mobility performance from non-ambulatory (Level 1: minimal active movement/fully dependent, and Level 2: requires physical assistance for transfers and limited ambulation/primary wheelchair use), household ambulators with severe fatigue (Level 3), community level ambulators with moderate (Level 4) and minimal fatigue (Level 5), to full motor function (Level 6). IMPROVE levels were assigned to our PMD cohort of 171 subjects, mean age (± SD) of 23.9 years ± 16.6, 60% female, with mtDNA (n=118, 69%) and nuclear (n=53, 31%) genetic etiologies who completed MM-COAST assessments. Mean (± SEM) MM-COAST scores in a prior MM Cohort (n=60) was 1.3 ± 0.1, n=53, with higher scores indicating greater disease severity¹. In this study (n=171), results demonstrate that most MM subjects were ambulatory with either mild (Level 5, n=39) or moderate (Level 4, n=72) fatigue. At IMPROVE Level 2, the MM-COAST scores were 2.15 ± 0.67 (n=18), Level 3 (1.69 ± 0.82, n=26), Level 4 (1.14 ± 0.99, n=72), Level 5 (0.65 ± 0.82, n=39), and Level 6 (-0.11 ± 0.73, n=14), p < 0.001, ANOVA. Kruskal-Wallis comparisons revealed significant differences between Levels 2 and 4 (p=0.0006), 3 and 5 (p=0.0003), 4 and 5 (p=0.04), and 4 and 6 (p=0.0003). Level 1 subjects were not included as they were too impaired to complete MM-COAST assessments. Higher MM disease severity was associated with lower mobility status by linear regression (β=-0.56, p< 0.0001, n=169). Further, IMPROVE levels correlated with MM-COAST key domains of strength: hip flexion (r=0.34, p<0.0001, n=129), ankle dorsiflexion (r=0.3, p=0.0008, n= 126), elbow flexion (r=0.3, p=0.0003, n=136); muscle fatigue: elbow flexion repetitions (r=0.33, p=0.004, n=73); balance: single leg eyes closed (r=0.39, p<0.0001, n=144), tandem stance eyes open (r=0.32, p=0.0002, n= 134) and eyes closed (r=0.32, p=0.0002, n= 129); nine-hole peg test (r=0.48, p<0.0001, n=134), functional dexterity test (r=0.4, p<0.0001, n=117); 30 second sit to stand (r=0.4, p<0.0001, n=104) and 6-minute walk test z-scores (r=0.58, p<0.0001, n=113). These correlations demonstrate that the IMPROVE system is clinically meaningful. In conclusion, our proposed IMPROVE classification facilitates subgroup analyses of MM subjects with similar mobility levels, which would transform cohort longitudinal data analyses. The IMPROVE may play a key role in future clinical trial design, subject selection, and longitudinal analyses of disease trajectories and assessment of treatment response.

Abstract #: 2023PA-0000000145

Presenter: Hatim Zariwala

Preservation of bioenergetics and inhibition of ferroptosis with the novel compound SBT-588 in Friedreich’s Ataxia cell models

Kropp LE, Handler A, Park Y, Redmon M, Zariwala HA*, Brown DA

Discovery Biology, Stealth BioTherapeutics, Inc., Needham, MA, USA

*Hatim.Zariwala@stealthbt.com

Friedreich’s ataxia (FA) is a monogenic disease caused by the loss of function of a mitochondrial protein called frataxin. The consequence is decline in mitochondrial respiration and cell death due to iron imbalance and accumulation. Here, we described a therapeutic approach using a novel molecule, SBT-588, that addresses both 1) dysregulated iron homeostasis that can trigger ferroptotic cell death cascades, and 2) disrupted bioenergetics due to dysfunctional mitochondrial respiration. SBT-588 was assessed in human fibroblast lines derived from FA patients (GM03665) and control (GM08402) individuals. We first assessed the cell protective effect of SBT-588 treatment against RSL3 and erastin, two known ferroptosis-inducing agents. RSL3 treatment resulted in significant reduction in cellular viability as measured by endogenous ATP levels. In patient and control cells, SBT-588 sustained cell viability at 111.4% and 84.5% compared to their untreated controls, respectively (p<0.05). A similar effect was observed using LDH assay to measure RSL3 induced cytotoxicity. SBT-588 significantly decreased cytotoxicity from 27.4% to 11.6% (p=0.0099). A challenge with erastin for 24 hours in the presence of ammonium citrate and ascorbic acid induced ferroptosis. In this assay, the ATP levels increased in a dose-dependent manner with SBT-588 treatment compared to controls. SBT-588 also decreased levels of 15-HETE, a metabolic by-product of lipoxygenase and marker of ferroptosis. 15-HETE is a potential biomarker for FA disease progression. In studies with RSL3, SBT-588 significantly reduced the concentration of 15-HETE in the supernatant from 970.38 pg/mL to 165.1 pg/mL after 4 hours (1-way ANOVA p=0.06). In affected cells, SBT-588 reduced 15-HETE concentration from 1587.92 pg/mL to 636.46 pg/mL in a trend towards significance (1-way ANOVA, n.s.). In a high-resolution respirometry study, SBT-588 was tested on its ability to restore cellular respiration following CI-inhibition with rotenone. A titratable increase in cellular respiration following SBT-588 treatment (EC₅₀=1.86 µM) was observed. Collectively, these data highlight the dual pharmacology of SBT-588 and indicate its potential as a therapeutic for FA.

Abstract #: 2023PA-0000000146

Presenter: Valerie Carosi

Behavioral Characterization Of MEPAN Mouse Model

Carosi V^1*, Beauplan M^1*, Murdock DG¹, Wallace, DC¹

¹Center for Mitochondrial and Epigenetic Medicine, Children’s Hospital of Philadelphia Research Institute, United States of America

murdockd@chop.edu

Mitochondrial Enoyl CoA Reductase Protein-Associated Neurodegeneration (MEPAN) is a rare genetic and neurological disease that causes childhood onset of dystonia, optic atrophy, and dysarthria. MEPAN is caused by mutations in the MECR gene that subsequently leads to deficiencies in the MECR protein, an important enzyme in mitochondrial fatty acid synthesis (mtFASII). Using CRISPR/Cas9 technology, we created the first MEPAN mouse model that contained patient specific mutations that reflect the symptomology of human MEPAN disease. We generated three compound heterozygous Mecr mutant mice: Mecr^285/285, Mecr^285/del10 and Mecr^285/del100. To better characterize the behavioral phenotypes of our MEPAN mouse model and to understand the pathophysiological basis of mtFASII deficiency diseases, we performed different behavioral analysis tests that assess gross locomotor activity, coordination, motor learning, gait, muscle strength and olfactory detection and differentiation between our three Mecr genotypes. Similar to MEPAN patients, MEPAN mice have a movement disorder as determined by rotarod, open field, and CatWalk analysis. The three genetically different mutant MEPAN mice have varying levels of motor dysfunction, with the Mecr 285/del10 having the most severe phenotype. Due to the strong expression of MECR in the olfactory bulb, we also tested the ability of the mice to smell using the olfactory habituation-dishabituation test. We found that the Mecr^285/del10 mice also had the most severe deficits in olfaction. These results confirm that our MEPAN mouse model recapitulates the prominent behavioral phenotypes of patients and can be used to screen therapeutics for MEPAN.

Abstract #: 2023PA-0000000147

Presenter: Fernando Scaglia

Expanded Clinical and Neuroradiological Phenotype of RARS2-Related Mitochondrial Disorder

Walimbe AS¹, Machol K², Kralik SF³, Mizerik EA², Emrick LT^{1, 2}, Scaglia F²*

¹Division of Pediatric Neurology and Developmental Neurosciences, Department of Pediatrics, Baylor College of Medicine, United States of America; ²Department of Molecular and Human Genetics, Baylor College of Medicine, United States of America; ³Department of Radiology, Baylor College of Medicine, United States of America

fscaglia@bcm.edu

RARS2-related mitochondrial disorder (MIM # 611523) is an autosomal recessive mitochondrial encephalopathy caused by biallelic pathogenic variants in the gene encoding the mitochondrial arginyl-transfer RNA synthetase (RARS2, NM_020320.5). RARS2 catalyzes the transfer of L-arginine to its cognate tRNA during the translation of mitochondrially-encoded proteins. The classical presentation of RARS2-related mitochondrial disorder includes pontocerebellar hypoplasia (PCH), profound developmental delay, feeding difficulties, and hypotonia. Most patients also develop epilepsy, which presents within the first three months of life with focal or generalized seizures that frequently become pharmacoresistant and lead to a developmental and epileptic encephalopathy (DEE). Here we describe a 4-year-old boy with developmental delay, hypotonia, and failure to thrive who developed an epileptic encephalopathy consistent with Lennox-Gastaut Syndrome (LGS), previously unreported in this disorder. He was noted to have dysmorphic features including bilateral macrotia, a depressed nasal bridge, down slanting palpebral fissures, and overriding second toes. Whole genome sequencing identified two variants in RARS2, c.36+1G>T, a previously unreported variant that is predicted to affect splicing, and is, therefore, likely pathogenic, and c.419 T>G, p.(Phe140Cys), a known pathogenic variant. Unlike most patients with RARS2-related mitochondrial disorder, our patient did not demonstrate PCH on brain MRI. Severe volume loss of the cerebral hemispheres and thinning of the corpus callosum were noted. Treatment with a ketogenic diet (KD) reduced seizure frequency and enabled him to attain developmental milestones. Our study highlights the importance of appropriate seizure phenotyping in RARS2-related mitochondrial disorder and indicates that patients can develop LGS, for which a KD may be a viable therapeutic option. This work further indicates that patients can present with dysmorphic features and may demonstrate other neuroradiological features instead of PCH on brain MRI. The case reported herein expands the known phenotypic spectrum associated with RARS2-related mitochondrial disorder and may help guide the diagnosis and management of patients with this condition.

Abstract #: 2023PA-0000000148

Presenter: Noa Sher

Characterization of mitochondrial augmentation at the single cell level

Noa Sher^1*, Niv Sabath¹, Patrick Maschmeyer^2,3, Igor Pozdnyakov¹, Hanan Khoury¹, Yehuda Brody¹, Tina Napso¹, Natalie Yivgi-Ohana¹, Caleb Lareau^4,5, Leif Ludwig^2,3

¹Minovia Therapeutics, Tirat Hacarmel, Israel; ²Berlin Institute of Health at Charité–Universitätsmedizin Berlin, Berlin, Germany; ³Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute for Medical Systems Biology (BIMSB), Berlin, Germany; ⁴Department of Pathology, Stanford University, Stanford, CA, USA; ⁵Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

*noa@minoviatx.com

Mitochondrial augmentation technology (MAT) enables cells with mitochondrial dysfunction to uptake clinical-grade isolated, healthy mitochondria. MNV-201, CD34+ hematopoietic stem and progenitor cells (HSPCs) augmented with allogeneic mitochondria from healthy placenta, is being developed for the treatment of patients with Pearson Syndrome, a fatal mitochondrial deletion syndrome.

A series of in vitro and in vivo studies using healthy augmented HSPCs from mobilized peripheral blood have demonstrated that the process of augmentation enables reconstitution of all major hematopoietic lineages, but no long-term persistence of exogenous mitochondrial DNA (mtDNA) was observed. Nonclinical data has demonstrated improved differentiation of HSPCs after mitochondrial augmentation in several models. In addition, patients treated in an open-label clinical trial or under compassionate use with a previous product, MNV-101, in which HSPCs were augmented with maternally derived syngeneic mitochondria, demonstrated improved quality of life and various systemic improvements. We therefore aimed to explore how augmentation of HSPCs may exert durable improvement in the hematopoietic compartment and whether persistence of exogenous mtDNA occurs in diseased cells.

To better understand the mechanism of action after MAT, we apply state-of-the-art single cell methodologies. Using the mitochondrial single-cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq), which enables sensitive detection of exogenous mtDNA at single cell resolution, we profiled 35,000 cells from augmented and control samples. We demonstrate that roughly 30% of the HSCs in the CD34+ population are augmented, suggesting a potential durable effect of the augmentation. We are currently using single-cell RNA-Seq to explore gene expression patterns associated with augmentation of HSPCs, both immediately after the augmentation process as well as after engraftment and differentiation in mouse models. We hypothesize that these findings will shed light on the mechanism by which MAT affects HSPCs and the derived hematopoietic cells.

Accumulating preclinical and clinical evidence suggests that immunological factors may play a more important role in non-hematopoietic primary mitochondrial disease (PMD) pathology than originally envisioned. It is our hope that an in-depth understanding of the effects of augmentation on HSPCs will enable the development of a product that can provide significant and durable benefits to patients with PMD.

Abstract #: 2023PA-0000000149

Presenter: Siddhesh Aras

Targeting MNRR1 as a therapeutic in models of mitochondrial dysfunction

Purandare N^1,2, Gomez-Lopez N^1,2, Fribley A³, Grossman LI^1,2, Aras S^1,2,*

¹Pregnancy Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health, and Human Services (NICHD/NIH/DHHS), Bethesda, MD 20892, Detroit, MI 48201, USA; ²Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA; ³Department of Pediatrics, and Department of Otolaryngology, Head and Neck Surgery, Wayne State University School of Medicine, Detroit, MI 48201, USA

*saras@wayne.edu (corresponding author)

The bi-organellar protein, MNRR1 (CHCHD2), is a key regulator of mitochondrial and cellular function. Reduced levels of MNRR1 are associated with a multitude of pathologies harboring a phenotype of mitochondrial dysfunction. In the mitochondria, MNRR1 functions towards activating OxPhos and also as an anti-apoptotic. In the nucleus, MNRR1 functions as a transcriptional regulator of stress-responsive genes. Enhancing the cellular levels of MNRR1 rescues the OxPhos defect in diseases such as MELAS, Niemann-Pick C1, and Amyotrophic lateral sclerosis. With respect to the upstream mechanism resulting in lower MNRR1 levels, using the MELAS heteroplasmy model, we have identified a HIF2α dependent transcriptional inhibition mechanism in cells with a 73% heteroplasmy. Genetic or pharmacologic inhibition of HIF2α rescues the MNRR1 levels along with the mitochondrial function. As a next step, we sought to identify specific activators of MNRR1 from and FDA-approved compound library with the potential to repurpose existing clinical drugs. One of the compounds identified on the screen and tested using orthogonal assays was Nitazoxanide, an anti-parasitic drug. We tested the efficacy of nitazoxanide in rescuing mitochondrial defect both in vitro and in vivo using the aforementioned models. Nitazoxanide treatment enhanced MNRR1 levels and enhanced mitochondrial function along with induction of the mitochondrial unfolded protein response (UPRmt) in all the model systems using cell lines and/or primary cells. As all these conditions have inflammation as an underlying pathology, for in vivo studies, we used a mouse model of intra-amniotic endotoxin induced placental inflammation resulting in pre-term birth. In support of our in vitro findings, MNRR1 levels were reduced in the placental tissues of endotoxin treated animals resulting in a significant reduction in gestational length along with over 90% fetal mortality. Oral administration of Nitazoxanide not only rescued the gestational defect but also reverted the enhanced mortality rate to baseline levels.

Abstract #: 2023PA-0000000150

Presenter: Kristin Edwards

Colonic Mitochondrial Dysfunction in Rodent Models of PCOS

Edwards*, K., Hoang, N.H., Rezq, S., Shawky, N.M., Brooks, K., Quin, R.M., Davenport, K., Yanes Cardozo, L.L., and Romero, D.G.

Department of Cell and Molecular Biology, The Women’s Health Research Center, Mississippi Center of Excellence in Perinatal Research, University of Mississippi Medical Center Jackson, MS, USA

*Corresponding author’s email: kedwards1@umc.edu

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is characterized by hyperandrogenemia and ovarian dysfunction. PCOS women exhibit low-grade systemic inflammation. Additionally, 42% of them often exhibit symptoms of irritable bowel syndrome (IBS) that is exacerbated by the diet. However, the underlying pathophysiology for the development of IBS is unknown. Mitochondrial dysfunction is associated with the development of inflammation and hyperandrogenemia can alter mitochondrial function. This study aims to investigate the mechanisms linking PCOS and mitochondrial dysfunction to the development of IBS symptoms. The hyperandrogenemic female (HAF) rodent models exhibit characteristics similar to PCOS women such as increased body weight and fat mass. At 4 weeks of age, female rats were implanted with dihydrotestosterone (DHT, 7.5mg/90 days) or placebo pellets. Rats were maintained on control diet (CD) or a western diet (WD: 43% carbohydrates mainly sucrose, and 40% fat). At 3 weeks of age, female mice were implanted with silastic tubes filled with DHT (8mg) or vehicle. Mice were maintained on either a low fat diet (LFD: 67.3 % carbohydrate, 4.3 % fat) or a high fat diet (HFD: 26% carbohydrates, 35% fat). At 15-22 weeks of age, colon tissues were collected for mitochondria isolation. Intact mitochondrial respiration was measured simultaneously with reactive oxygen species (mtROS) using the Oroboros O2k-FluoRespirometer. Quality of mitochondria was determined by the respiratory control ratio (RCR). Data was normalized to mitochondrial content using citrate synthase (CS) activity. Hyperandrogenemia in both HAF rats and mice caused a significant (p<0.05) decrease in complexes I- and II-driven respiration by 60% and 50%, respectively, compared to their respective normoandrogenemic controls. A significant (p<0.05) decrease in RCR was observed for both HAF rats (40%) and mice (20%) compared to their controls. Both HAF rats and mice showed a 2-fold increase in mtROS (p<0.05) compared to their respective controls. In control female rats, WD significantly decreased complex I- and II-driven respiration (50-54%, p<0.05), LCFA and MCFA oxidation (45% and 58%, respectively, p<0.05). In the HAF rats, WD further decreased complex I-driven respiration (30%, p<0.05), while all other respiration rates were decreased by 10%. Regardless of substrate provided, colon mtROS was significantly increased in both control (2.6-fold; p<0.05) and HAF rats (1.9-fold; p<0.05) on WD. HFD shows a trend for an increase in complex I-driven and complex II-driven respiration in control (122%) and HAF (174%) female mice. HFD diet showed no signifcant change in mtROS production. However, there is a slight decrease in mtROS in HAF mice on HFD (33%). The observed mitochondrial dysfunction in both rodent models of PCOS suggests that mitochondrial dysfunction may be involved in the development of IBS symptoms in hyperandrogenemic PCOS women. The WD exacerbated mitochondrial dysfunction in HAF rats, but on the other hand a HFD did not have the same effect on HAF mice. The modulatory effects of different diets and dietary components on colon mitochondrial function needs additional investigation. Further studies to re-establish normal colon mitochondrial function will provide options for IBS interventions where there are limited treatments.

Supported by NIH grants: P20PGM121334 (KE, SR, NMS, LLYC, DGR) ; AHA Career Development Award 938320 (NMS).

Abstract #: 2023PA-0000000151

Presenter: Hsin-Pin Lin

The OMA1-DELE1 mitochondrial integrated stress response is activated by diverse mitochondrial stressors to promote growth and survival in mitochondrial myopathy

Lin HP¹, Huang X¹, Gilsrud AJ¹, Li Y¹, Narendra DP*¹

¹Inherited Movement Disorders Unit, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA

*derek.narendra@nih.gov

Mitochondrial dysfunction triggers a strong mitochondrial integrated stress response (mt-ISR), which correlates with survival in early onset forms of mitochondrial myopathy (MM). Recently, it was shown that the mt-ISR can be activated through an OMA1-DELE1 signaling pathway, but whether and when the mt-ISR is protective in MM remains unclear. Here, we compare four transgenic mouse models of mitochondrial stress, including models of early (CHCHD10^G58R) and late (CHCHD10^S59L) onset MM and mild (CHCHD2/10 double knockout (KO)) and severe (TFAM muscle KO) oxidative phosphorylation (OXPHOS) deficiency, to define the mt-ISR in vivo. Strikingly, DELE1 activated the mt-ISR in all models but had the most remarkable effects on growth and survival in the two models with early mitochondrial stress. Notably, the mt-ISR did not directly improve the underlying OXPHOS impairment or associated reductive stress in these models but metabolically buffered the tissue by increasing tissue amino acids, nucleotides, and glycolysis intermediates. Thus, the mt-ISR may maintain growth despite mitochondrial dysfunction by rewiring the metabolome to bypass the OXPHOS deficiency. The mt-ISR was additionally responsible for upregulation of mitochondrial protease LONP1 and accounted for most of the transcriptionally driven remodeling of the mitochondrial proteome. This resolves that in mammalian striated muscle the mitochondrial unfolded response is a component of the DELE1 mt-ISR. Finally, we discovered that OMA1 and DELE1 have overlapping but separable effects on the mt-ISR and survival in MM. Indeed, neonatal survival in MM mice without OMA1 was associated with activation of an OMA1-independent mt-ISR. In summary, using four diverse mouse models of mitochondrial stress, we define the mt-ISR in striated muscle and identify a critical role for the mt-ISR in maintaining growth and survival in early onset MM.

Abstract #: 2023PA-0000000152

Presenter: Amel Karaa

Development and Validation of a Mitochondrial Disease-Specific Fatigue Questionnaire

Karaa A.¹*, Bahar R.², Clifford S.², Gorman G.³, Hansson M.J.⁴,

¹Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; ²Sprout Health Solutions, CA, USA; ³Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK; ⁴Abliva AB, Lund, Sweden

*akaraa@mgh.harvard.edu

Measuring the specific manifestations of fatigue in patients with primary mitochondrial disease (PMD) and understanding meaningful change in patients’ fatigue severity and frequency are fundamental for developing relevant and fit-for-purpose clinical outcome assessments for this patient population. The aim of this study was to understand the impact of fatigue on the daily lives of people with PMD and to develop a PMD-specific questionnaire for measuring fatigue in pivotal clinical trials.

Adults with genetically confirmed PMD participated in a 60-minute semi-structured concept elicitation (CE) interview exploring their experiences of fatigue. Transcripts were coded using a systematic coding process using MAXQDA™ software for qualitative data analysis. Themes were compared to existing fatigue questionnaires and item banks, and the PROMIS^® fatigue item bank was deemed most suitable in terms of breadth and relevance of PMD-related concepts. PMD symptoms and impacts identified in the CE interviews were mapped to items from the PROMIS^® fatigue item bank. The CE interview participants then participated in a 60-minute cognitive interview to assess understanding and relevance of the preliminary fatigue questionnaire items. Interview participants were recruited in the US in collaboration with the United Mitochondrial Disease Foundation (UMDF).

The CE interviews demonstrated that patients with PMD (N=12) experienced fatigue daily. Severity of fatigue ranged from moderate (significant impact on ability to perform daily activities) to severe (debilitating). The most commonly identified characteristics of PMD-related fatigue were physical tiredness, exhaustion, lack of energy, mental fatigue, and needing to lie down. Participants also reported that fatigue impacted several aspects of their lives, including ability to undertake housework, mood, leisure activities, physical activity, work/school, and social and family relationships. Based on how well the twenty mapped questionnaire items performed during the cognitive interviews, as well as the level of endorsement of the underlying concepts during the CE interviews, nine of the items were selected for the final PROMIS^® fatigue PMD short form questionnaire.

In conclusion, the PROMIS^® fatigue PMD short form items were found to be relevant, clear, and easy to answer by patients with PMD.

Abstract #: 2023PA-0000000153

Presenter: Christine Kong

Development of a semi-quantitative mitochondrial protein assay

Kong C*¹, Cahill A², Ganetzky R^^2,3

¹University of Pennsylvania College of Arts & Sciences, Philadelphia, PA, 19104, USA; ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA; ³Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

* = ckong12@sas.upenn.edu (presenter’s email); ganetzkyr@chop.edu (corresponding author’s email)

Mitochondria produce the majority of cellular ATP via oxidative phosphorylation. Mitochondrial DNA (mtDNA) encodes 13 polypeptides, which are translated by dedicated mitochondrial machinery. All 13 of these peptides are core oxidative phosphorylation subunits. With the rise of next generation sequencing, dozens of genetic diseases of mitochondrial translation have been identified. Many of these are not easily detected by conventional electron transport chain enzymology and no clinical assay directly assessing mitochondrial translation has been fully developed. We sought to develop a reproducible mitochondrial translation assay that was amenable to the clinical space. We used click chemistry to create a reproducible, semi-quantitative, non-radioactive translation assay in fibroblast cell lines. For assay development, we utilized one wild-type cell line, one cell line with a known translation defect (YARS2) and one cell line that had previously been found to have a translation defect with unknown genetic origin. Three biological replicates of each cell line were plated with methionine-free media. Emetine, a eukaryotic translation elongation inhibitor, was added to stop cytosolic translation. Then a “pulse” of alkalyne-containing methionine analogue, homopropargylglycine (HPG) was added for four hours before whole protein isolation and harvesting. Nascent polypeptides therefore incorporated HPG instead of methionine. HPG then “clicked” to an azide-functionalized fluorophore using a Click-iT® Chemistry Reaction Buffer Kit. Therefore, fluorescence intensity in each nascent mitochondrial peptide is proportional to its mitochondrial synthesis rate during the set reaction time. Proteins were then isolated and run on a sodium dodecyl sulfate-polyacrylamide gel. We observed a global decrease in fluorescence intensity in the bands of both subject cell lines, consistent with their known translation defects. However, interestingly, these two lines were distinct from each other: in the YARS2 mutant cell line there was consistently an accessory low molecular weight band, putatively correlating to mitochondrial tyrosyl-tRNA synthetase that was not present in any other cell line. We hypothesize that this corresponds to premature protein-truncation due to the relative unavailability of tyrosyl-tRNAs. Overall, we conclude that click chemistry can be utilized for a viable and sensitive semi-quantitative mitochondrial translation assay, but further testing is required to explore its sensitivity and ability to discriminate among different translation defects. One downside to this assay is that decreases in fluorescence intensity can be subtle and challenging to fully quantify. Our future aims include developing a more fully quantitative assay with internal standards for normalization. One possible avenue for that aim is to focus on a SILAC (stable isotope labeling by amino acids in cell culture) approach. Overall, we conclude that non-radiographic approaches to mitochondrial translation in accessible tissue (fibroblasts) can produce reproducible results and may be a viable direction for clinical assay development.

Abstract #: 2023PA-0000000155

Presenter: Gene Kelly

Elamipretide Demonstrates Target Engagement and Signs of Visual Function Benefits by Protecting Photoreceptors in Dry AMD

Abbruscato A¹, DiMatteo M¹, Kelly, G^1* On behalf of the ReCLAIM-2 investigators

¹Stealth BioTherapeutics Inc., Needham, MA, USA

The eyes are one of the largest consumers of ATP in the human body. Photoreceptors, the critical functional unit for vision, and the retina in general, are highly metabolically active thus requiring a high degree of mitochondrial function to meet this energy demand. It is estimated that the mitochondria within photoreceptors operate at 70 to 80 percent of their maximum capacity with very little reserve. Because photoreceptor cells have such a high metabolic rate with minimal functional reserve, these cells are especially vulnerable to mitochondrial dysfunction. Similar to what is observed in rare genetic mitochondrial diseases, mitochondrial dysfunction plays an integral role in the pathophysiology. Age-related macular degeneration (AMD), the leading cause of irreversible blindness in people ⩾55 years of age, is one such ophthalmic disease with mitochondrial origins. A reduction in photoreceptor function has been shown to be the root cause of vision loss of dry AMD and has also been observed in patients with rare genetic mitochondrial diseases. Furthermore, some abnormalities in the visual function testing of patients with genetic mitochondrial disorders have been shown to originate in the rod photoreceptors. Photoreceptor dysfunction can be quantified by changes in the ellipsoid zone (EZ), the mitochondrial-rich layer of the photoreceptors which can be visualized by optical coherence tomography (OCT). Measures of EZ integrity are recognized as biomarkers of retinal and mitochondrial health and were used as endpoints in the ReCLAIM-2 trial. This Phase 2 clinical trial evaluated the mitochondrial protective agent, elamipretide (ELAM), in patients with geographic atrophy (GA) secondary to dry AMD. Spectral domain OCT scans were analyzed using a machine learning–enhanced multilayer retinal segmentation platform to assess EZ integrity and OCT-based GA measurements. ELAM treatment was associated with a 43% reduction in the total loss of photoreceptor integrity (total EZ attenuation [tEZa], p=0.003), a demonstration of ELAM-mitochondria target engagement. In addition, more patients treated with ELAM experienced a ⩾ 2-line (⩾ 10 letter) gain in low-luminance best-corrected visual acuity (LL-BCVA, i.e. visual function, p=0.04) as compared to placebo. The change in tEZa over 48 weeks was significantly correlated with the change in LL-BCVA (p<0.0001). The ReCLAIM-2 findings support the use of EZ attenuation as a surrogate endpoint, predictive of disease progression, in clinical trials. As the field of ophthalmology evolves so too must the clinical endpoints. Historically, GA has served as the accepted surrogate endpoint in dry AMD. GA is not an appropriate endpoint to assess mitochondrial targeting drugs, such as ELAM, due to GA occurring after loss of the ELAM target, viable and functional photoreceptor mitochondria. Future studies on ELAM in dry AMD will utilize the newly accepted endpoint of EZ attenuation as a biomarker of mitochondrial health.

Abstract #: 2023PA-0000000156

Presenter: Jirat Chenbhanich

Inpatient Epidemiology, Healthcare Utilizaation And Comorbidities Of Melas: A National Inpatient Sample Study

Chenbhanich J^1*, Ungprasert P², and Kroner PT³

¹Department of Genetics and Genomic Sciences, Case Western Reserve University, Cleveland, OH, USA; ²Department of Rheumatic and Immunologic Diseases, Cleveland Clinic, Cleveland, OH, USA; ³Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Jacksonville, FL, USA

*jirat.chen@gmail.com

Previous studies have rarely reported on the prevalence, healthcare utilization, and comorbidities of patients with primary mitochondrial disorders in the inpatient setting. The use of the diagnostic code for Mitochondrial Disorders may lead to diagnostic code bias and pose challenges to generalizability due to disease heterogeneity. We investigated the clinical and economic aspects of hospitalized patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), a surrogate of primary mitochondrial disorders, using its specific diagnostic code. We identified patients with MELAS within the Nationwide Inpatient Sample (NIS) database from the year 2018 to 2020 using ICD-10 diagnostic code for MELAS (E88.41). This database prospectively collected data from over 4,000 hospitals across the United States. Data on patient and hospital characteristics, morbidities, mortality and expenditures were retrieved. A propensity-matched cohort of non-MELAS patients from the same database was constructed to serve as comparators. We identified 2,410 MELAS patients, corresponding to inpatient prevalence of 2.5 per 100,000 admissions. The most common reason for hospitalization was sepsis (22%), followed by seizure (20%) and stroke (17%). Compared to the comparators, MELAS patients had higher inpatient mortality (adjusted odds ratio [aOR] 2.76, 95% CI 1.64- 4.63) and various morbidities such as shock (aOR 3.24, 95% CI 2.23- 4.76), ICU admission (aOR 2.68, 95% CI 2.07- 3.48), and multi-organ failure (aOR 2.01, 95% CI 1.58- 2.56). They also had significantly higher odds of several comorbidities such as congestive heart failure, epilepsy, dementia, and gastroparesis. After adjusting for confounders, MELAS patients had longer length of stay (8.9 days vs. 4.8 days, p<0.01) and displayed a mean additional $15,325 (p<0.01) in total hospital costs and a mean additional $56,542 (p<0.01) in total hospitalization charges when compared to non-MELAS patients. We concluded that hospitalization of patients with MELAS was associated with a significantly higher morbidity, mortality, and expenditures compared to non-MELAS patients. Therefore, care of patients with MELAS should be focused on outpatient management to prevent hospitalization, and recommendations on effective inpatient management should be developed to reduce inpatient morbidity and mortality.

Abstract #: 2023PA-0000000157

Presenter: Andrew Sung

A deep mutational scanning framework for the clinical and functional characterization of OxPhos proteins

Sung AY^1,2*, Guerra RM³, Steenberge LH^2,4, and Pagliarini DJ³

¹Department of Biomolecular Chemistry, University of Wisconsin–Madison; ²University of Wisconsin School of Medicine and Public Health; ³Department of Cell Biology and Physiology, Washington University School of Medicine in St. Louis; ⁴Department of Biochemistry, University of Wisconsin–Madison

*andrew.sung@wisc.edu

Many mitochondrial disorders arise from mutations in poorly characterized proteins. Diagnosing these disorders is often difficult due to the uncertain effects these missense mutations have on protein function. To aid in disease diagnosis and accelerate the functional characterization of these proteins, we use deep mutational scanning (DMS) which assays the fitness of single-amino-acid substitutions in a protein of interest through a pooled growth competition. We first apply this methodology to a poorly characterized complex I (CI) assembly factor (AF), NDUFAF6, that has been linked to a wide spectrum of disorders ranging from pediatric encephalomyopathies to diseases of aging. Our data corroborate the handful of known NDUFAF6 pathogenic and benign missense variants and provide robust empirical evidence for the phenotypic annotation of nearly 40 variants of unknown significance and over 5,000 currently unannotated variants. These data also highlight regions of the protein that are poorly tolerant of substitutions, thereby suggesting functional relevance. These include an amphipathic C-terminal helix implicated in membrane binding and a patch on the protein surface that points to a protein-protein interaction as the main mode of action for NDUFAF6. Consistently, our cross-linking mass spectrometry and yeast two-hybrid assays identify the membrane-bound Q module subunit NDUFS8 as the interactor for NDUFAF6. Using native-PAGE western blots, we identify the stalled CI assembly intermediate upon loss of NDUFAF6, pinpointing the assembly step it mediates. Furthermore, we show that overexpression of NDUFS8 is able to complement knockout of NDUFAF6. Altogether, these data provide a comprehensive diagnostic resource for NDUFAF6-related diseases, support a model where NDUFAF6 mediates incorporation of NDUFS8 into CI, and suggest a potential avenue for therapeutic intervention. The systematic approach described here can be extended to other CI AFs and OxPhos proteins to evaluate variant pathogenicity, serve as a launching point for mechanistic investigations, and lay the groundwork for developing targeted therapeutics.

Abstract #: 2023PA-0000000160

Presenter: Jose Luis Marin Franco

Single-cell evaluation of bioenergetics by flow cytometry (e^-Flo)

Marin Franco JL^1*, Fuchs AL¹, Tarasenko T¹, Gordon-Lipkin EM¹, Kruk S¹, McGuire PJ¹.

Metabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD

* jose.marinfranco@nih.gov

Bioenergetic profiling has become an increasingly important tool for understanding the physiologic and pathologic processes involved in cancer and immune-mediated diseases. However, current methods of bioenergetic profiling have limitations including sample requirements, depth of investigation, accessibility, and reliance upon surrogate markers. To address these limitations, we developed a new method called e^-Flo. e^-Flo combines ATP fluorescence detection with the granularity of flow cytometry, allowing for the simultaneous study of bioenergetics and cellular identities/activation states across all cell types and species. One of the major advantages of e^-Flo is the ability to deconvolute bioenergetic profiles from complex populations of cells with minimal disruption. In C57bl/6J mice infected with influenza, metabolic profiles were determined for lung monocytes/macrophages, CD4⁺ T cells, and CD8⁺ T cells. In the absence of infection, monocytes/macrophages displayed roughly equal usage of FAO and OXPHOS for ATP generation, while CD4⁺ and CD8⁺ T cells showed predominance of OXPHOS with smaller contributions of FAO and glycolysis (N=20, p < 0.05; p < 0.0001). These bioenergetic profiles were consistent with resting immune cells. Following influenza infection, activated immune cells underwent metabolic reprogramming marked by a shift to Warburg-like metabolism. e^-Flo was used to determine the bioenergetic profile of activated immune cells ex vivo: monocytes/macrophages experienced a >60% increase in glycolytic activity for ATP production. OXPHOS was depressed to nearly undetectable levels, and FAO remained unchanged at low levels. CD4⁺ and CD8⁺ T cells showed similar bioenergetic profiles, with glycolysis being markedly increased while OXPHOS was mildly decreased. FAO in both CD4⁺ and CD8⁺ T cells was undetectable (N=20, p < 0.05; p < 0.0001). These bioenergetic profiles are consistent with activated cells, displaying an increased reliance upon glycolysis for ATP generation. Overall, e^-Flo is a powerful tool for defining bioenergetic profiles in complex cell populations. Furthermore, deeper phenotypic characteristics may be defined with additional cell surface markers. With its potential to provide high resolution bioenergetic profiling of disease processes, e^-Flo is poised to become an important tool for researchers in a variety of fields.

Abstract #: 2023PA-0000000163

Presenter: Jillian Jetmore

In Vitro System For Evaluating The Effect Of Mitochondrial Replacement On T Cell Phenotypes

Jetmore, J.¹, Marin Franco, J.¹, Tarasenko, T.¹, Kapnick, S.¹, McGuire, P.¹

¹Metabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MDjillian.jetmore@NIH.gov

Mitochondria are ubiquitous cellular organelles that are vital for several cellular processes such as homeostasis, metabolism, and bioenergetics. Mitochondria contain multiple copies of maternally inherited mitochondrial DNA (mtDNA). Pathogenic deleterious variants that cause mitochondrial disease (MtD) can occur in either the nuclear or mitochondrial genome, and patients with MtD present with heterogenous and multi-systemic disorders. While there are currently no treatments for MtD, recent in vitro and in vivo evidence supports mitochondrial augmentation therapy (MAT) as potentially capable of cellular metabolic reprogramming and rescue of mitochondrial function. One MAT strategy enriches a patient’s own hematopoietic and progenitor cells (HSPC’s) with healthy mitochondria isolated from healthy donors. While studies have demonstrated that reconstituting cells with exogenous, healthy mtDNA may rescue mitochondria functionality and bioenergetics, it is not well understood how certain mitochondrial sources may affect host cell immune phenotypes. Mitochondrial content, distribution and function vary in different cell types depending on their specific energy needs. A major question that remains is whether mitochondria isolated from non-immune organs with different bioenergetic requirements impart disparate cellular phenotypes in immune cells. To investigate the bioenergetic effect of various mtDNA donor sources on T cell phenotypes, we generated Rho0 cells from the Jurkat cell line, a human T cell line with an established history of elucidating various aspects of T cell function. mtDNA depletion was verified via qPCR and extracellular flux analysis. As expected, Rho0 clones are highly glycolytic when compared with their parent line. Via immunofluorescence imaging and electron micrographs, we confirmed that the Rho0 clones lacked nucleoid structures and had distorted mitochondrial morphology. After verifying mtDNA depletion, we performed polyethylene glycol fusion with platelets from a healthy donor. Successful fusion resulted in the growth and proliferation of trans-mitochondrial cybrids. Two weeks after mtDNA reconstitution, we observed partial restoration of OXPHOS; cybrids maintained a significantly lower oxygen consumption rate compared to the parent line. The turnover rates of respiratory chain proteins in human lymphoblastoid cells can range from several days to weeks. In future studies, we will follow cybrid OXPHOS over time to determine whether OXPHOS returns to parental levels. Further investigations will also address the effects of MAT sources on T cell phenotypes over time. This work has the potential to aid in the development of improved MAT and the restoration of mitochondrial function in immune cells.

Abstract #: 2023PA-0000000164

Presenter: Jeffrey Haltom

Mitochondria And Reproductive Biology

Jeffrey Haltom¹, Arrienne Butic¹, Dimitra Chalkia¹, Larry Singh¹, Olga Derbeneva¹, Masha Lvova¹, Patkik D. Wadhwa², Hyagriv Simham³, Douglas C. Wallace¹

¹Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104; ²Psychiatry & Human Behavior, University of California, Irvine, Irvine CA 92697; ³Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15213

The onset of puberty correlates with a wide spectrum of metabolic and degenerative diseases, and we have found that mice harboring the mtDNA ND6^P25L and COI^V421A missense mutations have a delayed onset of puberty. Moreover, pregnant ND6^P25L mice challenged with lipopolysaccharide deliver prematurely demonstrating that mitochondrial dysfunction can increase the risk of preterm birth (PTB). European American women harboring haplogroup H-HV mtDNAs have increased risk of PTB (OR ~ 2.7-3.0) while European American women with haplogroups J and T are protected (OR ~ 0.24). African American women, relative to European American women, have a reduced gestational age (-3.0 days, P = 0.01) and reduced birth weight (-211g, P < 0.0001). Hence, mtDNA variation may be an important factor in female reproductive outcomes. We next plan to examine the importance of African and Asian mtDNA haplogroups in PTB risk.

Abstract #: 2023PA-0000000165

Presenter: Laura Steenberge

Exploring new strategies for CoQ supplementation

Steenberge, LH ^{1,2, 3}* and Pagliarini, DJ⁴

¹Department of Biochemistry, University of Wisconsin – Madison, USA; ²Morgridge Institute for Research, USA; ³University of Wisconsin – Madison School of Medicine and Public Health, USA; ⁴Department of Cell Biology and Physiology, Washington University School of Medicine, USA

*steenberge@wisc.edu

Coenzyme Q (CoQ, ubiquinone) is a redox active lipid critical for cellular function. In addition to its central role in oxidative phosphorylation, it suppresses ferroptosis, enables uridine biosynthesis, and is a cofactor in myriad pathways. Primary defects in CoQ biosynthesis cause a variety of phenotypes, from nephropathy and myopathy to fatal multiorgan disease. Secondary defects in CoQ levels have been implicated in the pathophysiology of many common conditions, such as neurodegenerative diseases, cardiomyopathies, diabetes, and aging. Unfortunately, exogenous oral CoQ supplementation aimed at rectifying these conditions is difficult due to its large molecular weight and hydrophobicity. Only 2% of orally administered CoQ reaches the bloodstream and less than 15% of the CoQ that reaches tissues becomes incorporated into mitochondria. Here, we test less hydrophobic CoQ species with shorter isoprenoid tails to assess the minimum length needed for CoQ function. We establish that CoQ₄ can substitute for CoQ₁₀ in human cells, opening the door for CoQ₄ to be investigated as a more effective supplement for CoQ₁₀ deficiencies. In addition, we describe the synthesis and evaluation of an initial set of compounds designed to deliver CoQ in larger quantities selectively to mitochondria. These compounds incorporate a mitochondria-targeting triphenylphosphonium (TPP) moiety attached to CoQ through a reversible linker and are designed to accumulate in mitochondria and be cleaved to release native CoQ. Our results indicate that select versions of these compounds can successfully be delivered to mitochondria in a cell model and be cleaved to produced CoQ, laying the groundwork for further development.

Support/Funding

This work was supported by the NIH R35GM131795 (to D.J.P.), T32GM008505 (to L.H.S.) and T32GM140935 (to L.H.S), as well as Washington University School of Medicine in St. Louis, BJC Investigator Funds (to D.J.P.).

Abstract #: 2023PA-0000000166

Presenter: Cristina Remes

Therapeutic modeling in human fibroblast and C. elegans models of C12ORF65 primary mitochondrial disease

Lavorato M^1,2*, Haroon S^1,2, Elzanfali S¹, Mendel R¹, Remes C¹, Mathew ND¹, Nakamaru-Ogiso E^1,2, Falk MJ^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; ²University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104

*lavoratom@chop.edu (Corresponding author’s email)

C12ORF65-based primary mitochondrial disease (PMD) is an inherited genetic disorder characterized by impaired mitochondrial translation with disrupted mitochondrial energy production through oxidative phosphorylation. C12ORF65 disease patients develop progressive dysfunction of high-energy demand organs resulting in neuromuscular dysfunction, fatigue and exercise intolerance, neurodevelopmental disabilities, Leigh syndrome characterized by stress-induced episodic regression and metabolic strokes, peripheral neuropathy in the Charcot-Marie-Tooth spectrum, optic atrophy, sensorineural hearing loss, and/or metabolic instability with lactic acidosis. No FDA-approved therapies currently exist for C12ORF65 disease. To enable precision therapeutic development for C12ORF65 disease, we initiated a cross evolutionary species study in C. elegans and human fibroblast disease models. We obtained a primary human fibroblast cell line from a PMD proband who was homozygous for the c.210delA:p.G72AfsX13 pathogenic variant in C12ORF65. We analysed integrated mitochondrial respiratory capacity, respiratory chain (RC) enzyme activities, and OXPHOS protein levels. Celltiter-Glo® assay (Promega) was used to evaluate cell viability of proband and control fibroblasts under stress conditions and to test the efficacy of drug candidates for C12ORF65 disease. Mitochondrial morphology was analyzed in C12ORF65^-/- patient fibroblasts using fluorescence microscopy and MitoTracker Green live cell staining. A feeding RNAi knockdown model of c12orf65 was created in C. elegans, which was characterized at the levels of development, locomotor activity, and mitochondrial mass by feeding the c12or65 RNAi to the cox-4::GFP mitochondrial mass reporter C. elegans strain and utilizing the the BioSorter® platform (Union Biometrica). c12orf65(RNAi) C. elegans showed significant developmental delay, ~20% increased mitochondrial mass, and ~30% decreased thrashing activity as compared to wild-type control worms. RC enzyme activities analyses of C12ORF65^-/- patient fibroblasts showed 80% reduced CI enzyme activity, while mitochondrial respiratory capacity was marginally decreased in patient fibroblasts relative to healthy control. Hypersensitivity of several pharmacological or metabolic stressors and preliminary efficacy studies of 10 drug candidates were tested on patient cell viability. Results showed 50% decreased patient cell viability after incubation with 75 µM azide (complex IV inhibitor), while 25 nM rotenone (complex I inhibitor) decreased cell viability by 50% in both control and patient cells. Incubation of fibroblasts in glucose-free media showed a slight but significant reduction of patient cell viability relative to control. Among the drugs tested, only 20 mM dichloroacetate (DCA) increased cell viability in C12ORF65^-/- patient fibroblasts co-incubated in either 20 nM rotenone or glucose-free media. DCA also rescued mitochondrial morphology in rotenone-stressed C12ORF65^-/- cells.

With this study, we characterized key outcomes of C12ORF65^-/- deficiency relevant to human PMD both in human patient fibroblasts and in a multicellular invertebrate animal model, C. elegans. We were able to identify dichloroacetate as a putative drug candidate for C12ORF65 disease in human cell studies, with rescue of stressor-induced cell death and mitochondrial morphology. These c12orf65(RNAi) worm model offers a translational platform in which to validate the therapeutic efficacy of dichloroacetate and other drug candidates. Pursuing a “therapeutic cross-training” approach of candidate drugs in evolutionarily-diverse species will lead to the identification of safe and highly effective therapeutic leads for C12ORF65 disease to prioritize for clinical trial study in human patients.

Abstract #: 2023PA-0000000167

Presenter: Amel Karaa

Efficacy and safety of elamipretide in subjects with primary mitochondrial disease resulting from pathogenic nuclear DNA mutations (nPMD): phase 3 study design

*Karaa A¹; Mancuso M², on behalf on the SPIMM-301 Collaborative Network

¹Massachusetts General Hospital, Harvard Medical School Boston, MA, USA; ²Department of Clinical and Experimental Medicine, Neurological Institute, University of Pisa, Italy

*akaraa@mgh.harvard.edu

SPIMM-301 (MMPOWER-3) was a recently completed phase 3, randomized, double-blind, placebo-controlled clinical trial evaluating the use of elamipretide for the treatment of subjects with primary mitochondrial myopathy (PMM). Enrolled subjects had a variety of pathogenic variants in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) genes that caused their myopathy and the study did not meet the primary endpoints in the highly heterogeneous study cohort. However, post hoc genetic subgroup analyses demonstrated that patients with pathogenic nDNA variants experienced an improvement in 6MWT compared to placebo. This benefit was especially found in patients having mtDNA replisome-related mutations and chronic progressive external ophthalmoplegia (CPEO). Therefore, nuclear encoded control of mtDNA maintenance and replication may have particular relevance for elamipretide mechanism of action in this patient population. This finding, combined with the high unmet need for treatment in PMM patients, especially with respect to weakness and fatigue during activities, led to the development of SPIMD-301 (NCT05162768). SPIMD-301 (NuPower), a phase 3, randomized, double-blind, placebo-controlled clinical trial, is designed to evaluate the efficacy and tolerability of elamipretide in subjects with primary mitochondrial disease resulting from pathogenic nuclear DNA mutations (nPMD). The trial consists of a screening (⩽28 days), treatment (48 weeks), and follow-up period (4 weeks). The sponsor intends to provide Expanded Access for subjects who complete the study period through the Week 48 visit. Approximately 130 subjects (aged ⩾18 to ⩽ 70 years) with nPMD causing their myopathy will be randomized (1:1) to receive a single daily subcutaneous 60 mg dose of elamipretide or matching placebo for 48 weeks. The population will consist of 90 subjects who have nPMD associated with pathogenic mutations of the mitochondrial replisome ("replisome-related mutations") for primary analysis and an additional subset of up to 40 subjects who have nPMD associated with other non-replisome-related mutations. Subjects are required to have a diagnosis of nPMD with a predominant clinical manifestation of myopathy, which must include progressive external ophthalmoplegia (PEO) and exercise intolerance and/or skeletal muscle weakness. The primary efficacy endpoint will be distance walked (meters) on the 6MWT. Biomarkers and pharmacokinetic evaluations will also be explored. The study has been initiated, with first-site enrollment having occurred in April 2022. Further opportunities for enrollment continue in the US and Europe with an estimated completion date of June 2024. Results will help determine the efficacy and safety of mitochondrial-targeting therapy with elamipretide for the treatment of patients with nPMD. The complete SPIMM-301 study results, including the nDNA genetic mutation distribution, were recently accepted for publication and will be available soon in the journal Neurology.

Abstract #: 2023PA-0000000168

Presenter: Katherine Mitchell

Mitochondrial Dysfunction In Neuropsychiatric Disorders

Mitchell KL¹, Huang J¹, Morrow RM¹, O’Brien WT², Ciesielski B², Murdock DG¹, and Wallace DC^1,3

¹Center for Mitochondrial and Epigenomic Medicine, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, PA, 19104, USA; ²The Intellectual and Developmental Disorder Research Center, University of Pennsylvania and Children's Hospital of Philadelphia, PA, 19104, USA; ³Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

*wallaced1@email.chop.edu (*Corresponding author)

The brain is the tissue with the highest energy demand. Therefore, subtle changes in mitochondrial energy production should preferentially affect neurological function. To test this prediction, we created two C57BL/6 mouse lines harboring partial mitochondrial complex I defects due to missense mutations in the mitochondrial DNA (mtDNA), ND6^P25L and ND5^S204F. We then compared their behavior to that of normal C57BL/6 mtDNA mice, mtDNA^B6. Analysis of cortical respiration revealed that there was a sequential decline in respiration rate from mtDNA^B6 to ND5^S204F to ND6^P25L, with a maximum decrement in the ND6^P25L mice being reduced about 33% from the mtDNA^B6 respiration. We then subjected these mice to neuropsychological behavioral analyses. We used the Elevated Zero Maze to test anxiety and observed a striking stepwise increase in anxiety levels from mtDNA^B6 to ND5^S204F to ND6^P25L. To further test anxiety we used the Open Field paradigm and found that mtDNA^B6 and ND5^S204F mice were similar while ND6^P25L was significantly affected. Compulsive behavior was assessed by Marble Burying which revealed that ND5^S204F and ND6^P25L were similarly compulsive but different from mtDNA^B6. We have also recapitulated some known behavioral differences between male and female C57BL/6 mice and shown that some of these differences are not seen in ND5^S204F or ND6^P25L mutant mice. These behavioral studies show that mild mitochondrial defects can manifest classical neuropsychiatric symptoms. Hence, psychiatric disorders may be due to mitochondrial dysfunction.

Abstract #: 2023PA-0000000170

Presenter: Cristy Balcells

Patient co-creation and collaboration in thymidine kinase 2 deficiency (TK2d): Incorporating a stakeholder-led project steering committee into a qualitative observational study

Balcells C^1*, Waller K², Yeske P³, Hareendran A⁴

¹UCB Pharma, Smyrna, GA, USA; ²Lily Foundation, Warlingham, Surrey, UK; ³United Mitochondrial Disease Foundation, Pittsburgh, PA, USA; ⁴UCB Pharma, Slough, Berkshire, UK

*Cristy.Balcells@ucb.com

Thymidine kinase 2 deficiency (TK2d) is an ultra-rare mitochondrial disease associated with progressive, life-threatening, proximal myopathy that impacts feeding, breathing, and ambulation. Further insight into the global burden of illness and lived patient experiences is needed. In recent years, the value of collaboration with patients as key stakeholders at every stage of the drug development process has been recognized. However, in ultra-rare disease states, such as TK2d, where the patient community infrastructure is evolving, traditional evidence generation study methodologies often encounter limitations and fall short of engaging patient communities. In the design and development of a qualitative observational study of individuals affected by TK2d and their caregivers, a project steering committee comprised of TK2d patient community stakeholders was engaged to support the design, methodology, and execution of the study. While that study is in progress, here we report on the methodology and rationale to actively involve patient experts throughout the study process, and how to effectively include the unique insights and non-traditional research perspectives offered by TK2d patient stakeholders. These processes and methods will provide a model framework of best practices for patient engagement and co-creation of scientific evidence in ultra-rare communities. Capturing qualitative patient insights can provide a valuable complement to quantitative research data by improving all aspects of a study, including recruitment and accuracy and relevance of clinical research questions. This may also aid in the identification of relevant patient reported outcomes measures within mitochondrial disease research.

Abstract #: 2023PA-0000000171

Presenter: Cristy Balcells

Mapping mitochondrial disease global registries: A comprehensive review of publicly listed patient-driven and clinical umbrella registries related to mitochondrial disorders

Balcells C^1*, Housiaux S², and Mann K³

¹UCB Pharma, Smyrna, GA, USA; ²UCB Pharma, Brussels, Belgium; ³International Mito Patients, The Netherlands

*Cristy.Balcells@ucb.com

Mitochondrial diseases are a group of rare genetic disorders affecting multiple systems, including the central nervous system and skeletal muscles. They present with highly variable phenotypic and disease burden profiles which may exert significant morbidity and mortality. Low clinician awareness of rare diseases can delay diagnosis and patient access to novel therapies. Patient registries are valuable repositories of real-world scientific, clinical, and policy information relating to rare diseases, including mitochondrial diseases. Data from these registries can be utilized to support research and capture patient-reported outcomes. However, information may remain siloed within individual registries with differing structures, purposes, and data schema. Similarly, patients with mitochondrial diseases may belong to different support networks. In partnership with International Mito Patients, a global consortium of mitochondrial disease patient advocacy organizations, the need for improved sharing of information and opportunity for cohesion amongst various stakeholders interested in mitochondrial disease registries was recognized. Accordingly, global organizations and networks that host umbrella mitochondrial disease registries were surveyed to learn about their goals and the challenges they face. The aim of this report is to comprehensively describe the mitochondrial disease patient registry environment utilizing a web-based survey and additional online desk research. A questionnaire was developed to gather information through a series of open-ended, dichotomous, and multiple-choice questions that captured information on the registries’ management, content, demographic and health information collection, associated biorepositories, and geographical range. Registry management and content information collected included sources of funding, disease areas of focus, and number of patients. Demographic and health information collected included diagnostic status, symptoms, and treatments. Disease-specific registries were excluded from the analysis. Twenty-five global mitochondrial disease registries were identified for participation in the survey. Some organizations maintain more than 1 registry, resulting in manager overlap. Therefore, a total of 18 registry managers or teams were contacted, and information regarding more than 1 registry (where applicable) was requested. Responses were received from 15/18 registry managers (83%) regarding 19/25 registries (76%). Three of the remaining 6 registries were not contacted due to lack of available up to date contact information; survey responses were not received from the other 3 registry managers. Data from the survey responses and desk research are being evaluated and subsequently, a first-ever comprehensive overview of real-world information will be available to address an ongoing unmet need. For stakeholders, clinicians, and patients alike, these results will help facilitate future research and may aid in development of therapies for patients with rare and ultra-rare mitochondrial diseases.

Abstract #: 2023PA-0000000172

Presenter: Peter Stacpoole

Current Status of the Phase 3 Trial of Dichloroacetate (DCA) for Pyruvate Dehydrogenase Complex Deficiency (PDCD)

Stacpoole P.W.¹, Dorsey K.²

¹Department of Medicine, Professor, University of Florida, United States; ²Saol Therapeutics, Director of Clinical Operations, United States

pws@ufl.edu

Background: This is the first double-blind, placebo-controlled trial of any therapy for PDCD, a primary mitochondrial disease with phenotypic hallmarks including lactic acidosis and progressive neurological and neuromuscular degeneration. Approximately 90% of affected children harbor a loss-of-function mutation in the X-linked PDHA1 gene. The PDC is reversibly phosphorylated, and inactivated, by any of 4 differentially expressed isoforms of pyruvate dehydrogenase kinase (PDK). DCA is the prototypic pan-PDK inhibitor, a mode of action we postulate will stimulate residual PDC activity, thereby improving energy metabolism in PDCD individuals and, hence, their functionality. In 2018, we began a multicenter phase 3 trial of DCA in PDCD (NCT02616484) as the first such randomized, placebo-controlled trial (RCT) of any therapy for this disease.

Methods: Eligible children (6m-17y at entry) have a proven pathological mutation in any PDC component (except DLD) or in a pyruvate dehydrogenase phosphatase isoform, and a clinical phenotype consistent with PDCD. The trial is agnostic as to nutritional supplements or diet. A novel Observer-Reported Outcome (ObsRO) survey instrument was developed to quantify a subject’s at-home level of functionality, as reported by a parent/caregiver assessment of both severity and frequency of observations in seven domains identified as important by parents/caregivers: motor; breathing; seizures; eating; fatigue; sleep; and general health. Prior to randomization, subjects are genotyped to determine whether they are a “fast” (12.5mg/kg/12h) or “slow” (6.25mg/kg/12h) DCA metabolizer, based on haplotype variations in the gene encoding the glutathione transferase zeta 1isoform, which dechlorinates DCA to glyoxylate, which is inactive towards the PDC. The study design is a 9-month double-blind crossover comparison between oral DCA and placebo, during which the ObsRO is completed daily by a parent/caregiver. This is followed by an open-label phase of DCA administration. Primary endpoints evaluate efficacy outcomes during the double-blind period based on the ObsRO survey. Secondary endpoints evaluate Karnofsky/Lansky Performance Status.

Results: 34 children (8 males, 26 females), median age at entry 2 ½ yrs, have been enrolled from 9 sites across the United States, exceeding our original statistically based goal of 30 subjects. 33 patients have a mutation in PDHA1 and one patient has a mutation in PDHX. 27 patients were confirmed fast metabolizers and assigned the 12.5mg/kg/12hr dose level and 7 patients were confirmed slow metabolizers and assigned the 6.25mg/kg/12hr dose level. 23 patients have completed the double-blind period as of March 15, 2023, and 22 patients are now in the open-label phase. There have been no dropouts due to death or other reasons nor any Serious Adverse Events attributable to treatment. All but one patient who completed the double-blind period have continued on to open label DCA. Trial completion is expected in July 2023, with results to follow.

Conclusions: The potential innovation and impact of this trial are several-fold. Most importantly, it offers the first opportunity for the PDCD community to benefit from a targeted treatment of the disease. In addition, successful application of the ObsRO survey may have far-reaching consequences for the design and implementation of future interventional trials of mitochondrial and other rare disorders.

Abstract #: 2023PA-0000000173

Presenter: Elizabeth M. McCormick, MS, LCGC

Association of 37 mitochondrial DNA genes with Primary Mitochondrial Disease: Standardized assessment using the ClinGen Clinical Validity Framework

McCormick EM¹, Peterson JT¹, Taylor JP², Bluske K², Clause AR², Chandrasekhar A², Lowry J², Coffey AJ², Gai X^3,4, Falk MJ^1,5, Zolkipli-Cunningham Z^1,5, Rahman S⁶

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, USA; ²Illumina Clinical Services Laboratory, Illumina Inc., USA; ³Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, USA; ⁴Keck School of Medicine, University of Southern California, USA; ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA; ⁶Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, UK.

Primary mitochondrial disease (PMD) encompasses a broad group of phenotypically heterogenous disorders caused by impaired mitochondrial energy metabolism. PMD is caused by pathogenic variants in either nuclear or mitochondrial DNA (mtDNA), where each of the 37 mtDNA genes has been reported to be associated with PMD. The ClinGen Mitochondrial Disease Gene Curation Expert Panel (GCEP, Mitochondrial Diseases Gene Curation Expert Panel - ClinGen | Clinical Genome Resource), funded since 2017 through the National Institutes of Health (NIH) National Institute of Child Health and Human Development (NICHD) U24 grant program, has been a highly productive effort engaging more than 50 international PMD experts. The first three-year project period focused on systematic expert panel evaluation of the strength of evidence between select nuclear and mtDNA genes and Leigh syndrome spectrum (LSS).

Re-funded in 2021 by the NICHD together with the National Institute of Neurological Disorders and Stroke (NINDS), the Mitochondrial Disease GCEP focus has expanded to address the larger imperative to perform rigorous expert panel curation of all genes associated with PMD. Mitochondrial Disease GCEP curation of the 37 mtDNA genes for association with PMD was prioritized and is being performed according to the ClinGen Gene Clinical Validity Curation Process by eight biocurators. Baseline inclusion criteria for scoring variants in reported cases have been refined, along with approaches to scoring variants with varying levels of functional validation, including cybrid cell lines and single fiber studies. This standardized framework allows for systematic and rigorous review of published literature to reach consensus on the strength of the relationship between each gene and PMD. In a second step, the biocurators’ gene-disease association report and recommendation are reviewed and a final assessment agreed upon by the international panel of PMD experts.

The Mitochondrial Disease GCEP has completed curation of 15 mtDNA genes for their relationship with PMD, with completion of curation for the remaining 22 mtDNA genes anticipated by late spring 2023. Among the completed curations, 9 genes reached a ‘Definitive’ classification, 3 were classified as having a “Moderate” association, and 3 were found to have a “Limited” association with PMD. Overall, this work has clarified the varied strength of mtDNA gene relationship to PMD based on published literature evidence. This collaborative international effort remains actively underway to rigorously review gene-disease relationships for PMD, which is critical for accurate variant interpretation and confirmation of genetic diagnoses necessary to optimize medication management, tailored multi-system organ screening, accurate recurrence risk counseling and prevention, and clinical trial inclusion.

Abstract#: 2023PA-0000000174

Presenter: Elizabeth M. McCormick, MS, LCGC

Application of mitochondrial DNA variant interpretation specification guidelines to 96 MITOMAP-Confirmed disease-associated variants: Outcomes and identification of strengths and limitations

McCormick EM¹, Lott MT², Muraresku C¹, Wong S³, Procaccio V⁴, Wallace DC^2,5, Gai X^6,7, Falk MJ^1,5

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA; ²Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, USA; ³Ambry Genetics, USA, ⁴Biochemistry and Genetics Department, MitoVasc Institute, France; ⁵Perelman School of Medicine, University of Pennsylvania, USA; ⁶Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, USA; ⁷Keck School of Medicine, University of Southern California, USA.

Mitochondrial DNA (mtDNA) variant pathogenicity assessment requires special considerations given certain features of the mtDNA genome, including variant heteroplasmy, threshold effect, absence of splicing, presence of haplogroups, and maternal inheritance. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium and obtained Expert Panel status (Mitochondrial Disease Variant Curation Expert Panel, or Mito VCEP) from the Clinical Genome Resource (ClinGen). The ClinGen Mito VCEP, funded in part beginning in 2016 through ClinGen and further in 2017 through the National Institutes of Health (NIH) National Institute of Child Health and Human Development (NICHD) U24 grant program, has been a highly productive effort and has engaged more than 50 international experts (https://clinicalgenome.org/affiliation/50027). The first project period focused on specifying variant interpretation guidelines for select nuclear genes and mitochondrial DNA genes. After critically reviewing the 2015 American College of Medical Genetics (ACMG) and Association of Molecular Pathology (AMP) standards and guidelines widely used for clinical interpretation of DNA sequence variants, further specifications and additional guidance for mtDNA variant classification were provided and published (Richards et al., 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544753; McCormick et al., 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717623). Issues addressed included mtDNA genome composition and structure, haplogroups and phylogeny, mtDNA genomic databases and computational algorithms, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA.

Re-funded in 2021 by the NICHD together with the National Institute of Neurological Disorders and Stroke (NINDS), the Mito VCEP is now focused solely on mtDNA variant curation according to the mtDNA variant interpretation specifications published by this group. The two step-review, first by one of four biocurators and then by an international panel of mitochondrial experts, is prioritizing variant curation for the 96 mtDNA variants listed as “Confirmed” in MITOMAP, as these variants are widely considered to be the “gold standard” variants associated with primary mitochondrial disease by clinicians and researchers. MITOMAP variant curation differs from the ACMG/AMP classification system in several aspects, including that MITOMAP curation has largely been performed by a single manual curator (MTL) reviewing all published literature, followed by review from the heads of the MITOMAP clinical team (VP, DCW).

The Mito VCEP meets twice monthly to review mtDNA variant curations, where curation of 85/96 variants have been completed, with anticipated curation over the next month of the final 11 variants. Interestingly, after curation according to the ACMG/AMP classification system specified for mtDNA variants, 20 MITOMAP “Confirmed” variants were classified as pathogenic, 45 as likely pathogenic, and 20 as of uncertain significance. The Mito VCEP elected to modify the final classification of 14 of the 85 variants whose review has been completed (of the 45 variants classified as likely pathogenic, 9 were upgraded to likely pathogenic from uncertain significance; of the 20 variants classified as pathogenic, 5 were upgraded from likely pathogenic to pathogenic), highlighting the limitations to the current ACMG/AMP classification system specified for mtDNA variants. Overall, rigorous and systematic curation of the “gold standard” set of mtDNA variants associated with primary mitochondrial disease has highlighted strengths and limitations to the current mtDNA variant specifications. We will use this review as the basis to propose revisions to the ACMG/AMP mtDNA variant specifications initially published by this VCEP.

Abstract #: 2023PA-0000000175

Presenter: Piotr Kopinski

Uveal melanoma cells exhibit variable growth patterns and distinct mitochondrial respiration profile compared with primary human uveal melanocytes

Kopinski, PK, Ligezka AN, Erickson S, Morava E, Kozicz T, Dalvin, LA*

Mayo Clinic, Rochester, MN, USA

dalvin.lauren@mayo.edu

Uveal melanoma is a deadly cancer with high metastatic potential. Mitochondrial metabolism and epigenetics are known to play a role in tumorigenesis, progression, and metastasis. However, primary human uveal melanocytes are difficult to obtain, and the metabolic state of malignant versus benign uveal melanocytes has not been well characterized. We isolated primary human uveal melanocytes from six donor eyes and successfully propagated them in 2D cell culture. We then determined primary uveal melanocyte morphology, growth curves and respiratory states using Seahorse and compared them with two uveal melanoma cell lines, MP46 (highly adherent), and MP65 (partially suspended). In 2D cell culture, uveal melanoma MP46 and MP65 cell lines formed primitive colonies with light to moderate pigmentation, while primary uveal melanocytes formed higher order patterns with heavy pigmentation. In addition, uveal melanoma MP46 cells grew faster than PK3 and PK4 benign melanocytes. Metabolically, uveal melanoma MP46 cells showed a glycolytic metabolic profile with statistically significant decrease in basal respiration, increase in proton leak respiration, and decrease in coupling efficiency when compared with PK4 primary uveal melanocytes. Uveal melanoma cell lines showed distinct morphological, physiologic, and metabolic characteristics compared with benign uveal melanocytes. Interestingly, despite increased growth rate, MP46 uveal melanoma cells showed decreased coupling efficiency compared with PK4 primary uveal melanocytes. Thus, mitochondrial function may underlie distinct characteristics of primary uveal melanocytes and uveal melanoma cell lines.

Abstract #: 2023PA-0000000176

Presenter: Gunjan Purohit

LACTB deletion alters mitochondrial metabolism and impacts intermembrane contact sites

Purohit Gunjan^1*, Khalimonchuk Oleh ^1,2,3

¹Department of Biochemistry, ²Nebraska Redox Biology Center, University of Nebraska, Lincoln, NE, ³Fred and Pamela Buffett Cancer Center, Omaha, NE

*gpurohit2@unl.edu (okhalimonchuk2@unl.edu)

Mitochondria are crucial regulators of cellular physiology, but many aspects of their functions, including roles of many conserved mitochondrial proteins remain incompletely understood. The mitochondrial protease LACTB is a homolog of the penicillin-binding protein found in Gram-negative bacteria. This serine protease is localized in the mitochondrial intermembrane space where it forms long filamentous structures, the significance of which is unknown. The protease is postulated to support membrane organization and metabolic regulation and influence mitochondrial lipid metabolism, thereby acting as a tumor suppressor in breast cancer, but its exact physiological role remains obscure. Here, we generated a LACTB-knockout cell line to investigate its role in mitochondrial homeostasis and ultrastructure. Our data indicate that whereas the proliferation and doubling rate of cells lacking the enzyme remain similar to that of wild-type cells, the migratory properties of LACTB-deleted cells are altered. Moreover, LACTB-devoid cells exhibit reduced mitochondrial respiration as well as ultrastructural alterations in response to homeostatic challenges. Consistent with these findings, LACTB depletion upregulates the constituents of the mitochondrial contact site and cristae organizing system (MICOS) complex, suggesting the enzyme’s role in regulation of contacts between the inner and outer mitochondrial membranes. Overall, our findings provide new insights into LACTB’s role in regulating physiological outputs related to mitochondrial membrane remodelling.

Abstract #: 2023PA-0000000177

Presenter: Lishuang Shen

MSeqDR Virtual Registry of 7,500 Leigh Syndrome and Primary Mitochondrial Disease Cases Constructed through Semi-Automated Literature Mining and Expert Curation

Lishuang Shen^1,7, Marie T. Lott², Elizabeth M. McCormick³, Colleen C. Muraresku³, Kierstin Keller², ClinGen Mitochondrial Diseases Gene Curation Expert Panel, Douglas C. Wallace ^2,3,4, Zarazuela Zolkipli-Cunningham^2,3,4, Shamima Rahman⁵, Marni J. Falk^2,3,4, Xiaowu Gai^1,6

¹Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA; ²Center for Mitochondrial and Epigenomic Medicine, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA; ³ Mitochondrial Medicine Frontier Program, Children’s Hospital of Philadelphia, Philadelphia, PA; ⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; ⁵Department of Genetics & Genomic Medicine, UCL Great Ormond Street Institute of Child Health, London, UK; ⁶Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA; ⁷Clinical Department, Prenetics Limited, Hong Kong SAR

Primary mitochondrial disease (PMD) has a low prevalence of 1 in 4,300 people and the most common PMD is Leigh Syndrome (LS), with a prevalence of 1 in 34,000 people. PMD community registry creation requires intensive collaborative effort thus most registries have limited local or national cases. To enhance our knowledge of PMD, we built within MSeqDR a PMD Virtual Registry using both semi-automated literature mining and expert review. Specifically, we developed a semi-automated pseudo-case curation platform, hosted at MSeqDR.org (https://mseqdr.org/virtualregistry.php), to aid in the capture, extraction, and standardization of case-level data from publications by manual review. In parallel, the NIH-funded ClinGen Mitochondrial Diseases Gene Curation Expert Panel (HD-093483, https://www.clinicalgenome.org/affiliation/40027/, ) manually curated ~350 deeply-phenotyped cases associated with 113 genes from literature to evaluate the causative gene associations for Leigh Syndrome spectrum (LSS) disorder. The heterogeneous clinical and demographic data were mapped to over 100 “standard” clinical, biochemical, and genetic feature terms including Human Phenotype Ontology (HPO) and OMIM disease terms. Case-level data was divided into over 272,000 atomized feature records. The Web front end includes a Case Browser for queries using single or composite filters constructed from atomized and standardized keywords such as OMIM disease, (causative) genes and variants, phenotypes and HPO terms, inheritance mode, zygosity, consanguinity, sex, age at onset and death, ethnicity, PubMed ID and title. Matching cases are hyperlinked to the single case full report and curation pages, which presents all data elements of a case in both standardized and original terms. Registered MSeqDR users can curate phenotype information in crowdsource fashion to collaboratively build this community resource. Each user’s curations are saved and tracked, without overwriting other contributors’’ input. As of March 2023, the MSeqDR PMD Virtual Registry collection contains 7,550 de-identified PMD pseudo-cases which exceeds most existing ad-hoc LS/LSS or PMD case registries. It contains ~1,150 LS/LLS cases (>800 from semiautomatic literature mining plus 350 from U24 project expert curation) plus ~3,000 other PMD cases. In addition, over 3,000 virtual cases from MitoPhen were downloaded and transformed for use in the Virtual Registry. Cases from 586 genes are included -, of which 126 genes have been published in our Mitochondrial Disease Gene Compendium Edition 1 and 44 genes are being curated for Edition 2, thus reflecting the relevance of the collected data to PMD. 6933 cases carry from one to over 10 of the 8994 phenotypes, where 1365 cases have at least 5 phenotypes reported, reflecting the phenotypic richness and need for further standardization efforts. 5210 cases carry one of the 2099 pathogenicity-assessed mutations whereas 3916 of them carry a 2nd mutation. None carry a 3rd valid mutation. Inheritance modes for nearly 6,000 cases are available, including 3,699 cases with mitochondrial (HP:0001427), 498 for autosomal recessive (HP:0000007), 314 with autosomal dominant (HP:0000006), and 104 with X-linked (HP:0001417) inheritance. The most common phenotypes are skeletal muscle atrophy (HP:0003202), increased serum lactate (HP:0002151), and abnormal basal ganglia morphology (HP:0002134). Full case curation and in-depth stratification and correlation analysis remain ongoing.

Abstract #: 2023PA-0000000178

Presenter: Magnus Hansson

FALCON: A Randomized, Placebo-Controlled Study of the Efficacy of KL1333 in Adult Patients with Primary Mitochondrial Disease

Hansson M.J.*, Ence F., Nesse D., Elmér E., and Donnelly E.

Abliva AB, Lund, Sweden

*magnus.hansson@abliva.com

The FALCON study, a global, placebo-controlled and potentially registrational Phase 2 study of KL1333 in adult patients with primary mitochondrial disease commenced in December 2022. The primary objectives of the FALCON study are to evaluate the efficacy of KL1333 versus placebo on fatigue symptoms and impacts on daily living, as well as on functional lower extremity strength and endurance. The efficacy of KL1333 on these debilitating disease manifestations of primary mitochondrial disease (PMD) will be evaluated over 48 weeks of treatment. The study will also evaluate the safety and tolerability of KL1333.

KL1333 aims to restore the balance of the essential coenzymes NAD⁺ and NADH, thereby improving energy metabolism and stimulating the build-up of new mitochondria. In phase 1 studies, KL1333 was safe and well tolerated. In a phase 1b PMD patient cohort, there were signals of improvement of fatigue and muscle function in KL1333-treated patients versus those treated with placebo. In addition, there was a correlation between exposure and efficacy, and target engagement was demonstrated.

The FALCON study is actively recruiting adult PMD patients suffering from fatigue and myopathy in both the US and Europe. An interim analysis, conducted on up to 40 patients treated with KL1333 for six months, will be conducted in 2024 to determine the final study size. The study is registered on clinicaltrials.gov, NCT05650229.

Abstract #: 2023PA-0000000179

Presenter: Sonal Sharma

Identifying Neuroimaging Patterns in Mitochondrial Leukoencephalopathies

Sharma S*^1,2, Peterson J², Alves C³, Goldstein A²

¹Division of Neurology, ²Mitochondrial Medicine Frontier Program, Division of Genetics, ³Division of Radiology, Children's Hospital of Philadelphia, Philadelphia

*sharmas10@chop.edu

Objective: To identify patterns of white matter involvement in mitochondrial disease.

Background: Mitochondrial disease has been estimated to affect 1 in 4,300 individuals. Primary mitochondrial diseases (PMD) are caused by pathogenic variants in >300 genes. PMD can present at any age with an average of 16 multi-systemic symptoms per patient and often involve the neurological system due to its high metabolic demand. Incidence of leukoencephalopathies is unclear and ranges widely from 1 in 50,000 to 1 in 7663. Studies have reported variable incidence of idiopathic leukoencephalopathies ranging from 5.8% to 58%. There is limited knowledge regarding mitochondrial leukoencephalopathies. In this study, we reviewed patterns of white matter involvement on brain MRI in patients with genetically-confirmed PMD.

Design/Methods: Retrospective chart review of 186 patients with genetically-confirmed PMD was conducted at the Children’s Hospital of Philadelphia in the Mitochondrial Medicine Frontier Program (MMFP). Clinical cases were identified from the MMFP, IRB-approved CHOP Study #08–6177 (M.J.F., Principal Investigator), and by the electronic medical record (EMR) database. Brain MRI studies available for 153 patients were analyzed to identify the presence of white matter involvement. Neuroimaging patterns were delineated based on location, type of involvement (leukodystrophy vs hypomyelination), T1 and T2 appearance, cystic changes, volume loss, atrophy, restricted diffusion, contrast enhancement, and associated brain structure involvement. Information was also collected regarding CSF protein level, plasma lactate level, and lactate peak on magnetic resonance spectroscopy imaging.

Results: White matter involvement was observed in 61 out of 153 patients with MRI studies available for review. Genetic diagnosis of these patients included genes involved in mitochondrial translation (C12ORF65, MRPS34, RMND1, EARS2, CARS2, FARS2, VARS2), mtDNA depletion (FBXL4), oxidative phosphorylation (AIFM1), complex I function (NDUFS1, NUBPL), complex II function (SDHB), complex IV function (SURF1), single large scale mtDNA deletion (SLSMD), mt-tRNAs involved in mitochondrial translation (MT-TL1), pyruvate metabolism (PDHA1), valine catabolism / secondary PDH deficiency (ECHS1) and thiamine transport (SLC19A3). A leukodystrophy pattern was seen in 14 patients, while hypomyelination was observed in 16 patients, the remaining were thought to fit a delayed myelination pattern. Location was periventricular in 23, diffuse in 15 and involving corpus callosum in 25 patients. Restricted diffusion was observed in 18, cystic changes in 10, volume loss in 14 and associated cerebral/cerebellar atrophy in 11 patients.

Conclusions: Leukoencephalopathy with specific imaging patterns in neurologically complex patients with multisystemic presentations is consistent with the potential diagnosis of PMD and should warrant detailed diagnostic evaluation for mitochondrial disease. We believe this will help guide clinicians to develop a higher level of suspicion of mitochondrial disease in patients presenting with leukoencephalopathy and allow for pattern recognition in relation to specific genes.

Abstract #: 2023PA-0000000180

Presenter: Cristina Remes

Characterization of 19 mitochondrial aminoacyl-tRNA synthetases in C. elegans

Remes C¹, Matthew ND.¹, Schrope S¹, Nakamaru-Ogiso E^1,2, Falk MJ^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; ²Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

Mitochondrial aminoacyl-tRNA synthetases (mt-ARS) are essential components of the mitochondrial translation machinery that catalyze charging of mitochondrial transfer RNAs (tRNAs) with their cognate amino acid. Although mt-ARS have a common biochemical function, patients with mt-ARS mutations present neurological disorders with a wide range of clinical phenotypes, severity, and age of onset. To systematically characterize the function of each mt-ARS, we knocked down the full set of 19 conserved mt-ARS genes using feeding RNAi interference (RNAi) in the C. elegans invertebrate (worm) model organism. Individual silencing of the 19 mt-ARS genes in C. elegans was achieved by feedingRNAi specific for each mt-ARS. We assessed the development of C. elegans knockdown strains by measuring worm length at larval stage L4. To quantify the degree of in vivo mitochondrial unfolded protein response (UPR^mt) stress, we used C. elegans carrying hsp6p::GFP reporter to measure the increase in GFP fluorescence upon knockdown of each mt-ARS gene. Worm activity was also analyzed using a thrashing assay to quantify body bends per second when worms were swimming in liquid media. Our results showed that inactivation of every mt-ARS gene caused an approximately 20% decrease in worm length at stage L4, suggesting development was delayed in the knockdown strains. Distinct levels of UPR^mt stress induction occurred upon inactivation of each mt-ARS gene, with a set of mt-ARS genes which activated and another set of mt-ARS genes having only minimal effect on the UPR^mt stress response. Among the 19 mt-ARS genes, we observed the strongest activation of UPR^mt stress response in the mitochondrial aspartyl-tRNA synthetase (DARS2) knockdown worm strain, with approximately 10-fold higher level of hsp6p::GFP fluorescence compared to the other screened mt-ARS knockdown strains. In our study, we characterized the relative effects of loss of function in each of the 19 mt-ARS genes on animal development, UPR^mt stress induction, and animal activity in C. elegans. Our results suggest that a consistent degree of developmental delay with variable degrees of UPR^mt stress response occurred upon knockdown of individual mt-ARS genes. Future experiments will characterize of the effects of CRISPR/Cas9-based knock-out and/or known human disease pathogenic variant knock-in models of mt-ARS genes. Ultimately, these analyses will help to identify common pathophysiologic mechanisms across the broad class of mt-ARS-based primary mitochondrial disease.

Abstract #: 2023PA-0000000181

Presenter: Ryan Morrow

Deficits in mitochondrial oxidative phosphorylation enhance SARS-CoV-2 replication

Soto Albrecht YE^1-4, Morrow R^1*, Olali A¹, Kenney D^3,4, Potluri P¹, Murdock DG¹, Angelin A¹, Douam F^3,4, Wallace DC^1,5

¹Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA; ²Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; ³Department of Microbiology, Boston University School of Medicine, Boston, MA; ⁴National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA; ⁵Division of Human Genetics, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

*morrowr1@chop.edu (*Corresponding author’s email)

SARS-CoV-2 rewires host metabolism to optimize virus production. While glycolysis is necessary for virus production, whether mitochondrial oxidative phosphorylation (OXPHOS) is required for SARS-CoV-2 replication is unknown. Mitochondrial DNA (mtDNA) codes for 13 critical oxidative phosphorylation (OXPHOS) polypeptides of the electron transport chain (ETC) as well as the mitochondrial translation machinery necessary for their production. We discovered ~5 to 100-fold greater SARS-CoV-2 virus production in infected human ACE2-expressing A549 cells deficient in OXPHOS due to mtDNA depletion (ρ⁰ cells). A similar infection enhancement effect was observed upon blocking mitochondrial translation and chemically inhibiting ETC complexes. Cells deficient in OXPHOS demonstrated increased size and distribution of viral replication centers and promoted infectious viral particle release two hours earlier than WT cells following infection. Notably, the interferon-stimulated gene response at the transcript and protein levels remained intact throughout infection. Reintroduction of mtDNA in the ρ0 cells reinstated OXPHOS and impaired SARS-CoV-2 viral replication compared to parental ρ⁰ cells. In summary, our findings support that endogenous mtDNA expression and OXPHOS function exert an antiviral effect on SARS-CoV-2 infection. Work is ongoing to explore how mitochondrial OXPHOS regulates the SARS-CoV-2 life cycle and the cytosolic mediators of this relationship. We hope to inform future approaches to modulating mitochondrial function in infected cells, animal models, and humans to mitigate severe sequelae of COVID-19.

Abstract #: 2023PA-0000000182

Presenter: Shannon Schrope

High-throughput pre-clinical evaluation of candidate therapies using RNA interference of DLD-based Primary Mitochondrial Disease in Caenorhabditis elegans

Shannon Schrope¹(schropes@chop.edu), Kelsey Keith³ (keithk@chop.edu), Ryan Mendel ¹(mendelr@chop.edu), Suraiya Haroon ^1,2(haroons@chop.edu), Prabhjot Kaur ¹, Eiko Nakamaru-Ogiso^1,2(ogisoe@chop.edu), Neal D. Mathew¹(mathewn1@chop.edu) and Marni J. Falk^1,2(falkm@chop.edu)

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia. ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. ³Department of Biomedical & Health Health Informatics (DBHi), The Children's Hospital of Philadelphia

Introduction: Dihydrolipoamide dehydrogenase (DLD) deficiency is a rare, autosomal recessive, genetic disorder caused by pathogenic variants in DLD for which there are no FDA approved therapies. Our research group seeks to identify effective therapies for DLD disease using pre-clinical translational animal models. Here, we describe a novel high-throughput screening (HTS) approach we have developed to accelerate discoveries in a liquid feeding RNA interference (RNAi) gene knockdown model of DLD deficiency in the invertebrate animal model, C. elegans (microscopic worms).

Methods: We developed a customized HTS platform for high content imaging (Cellinsight CX5, Thermo Scientific) in C. elegans to screen drug libraries for candidate small molecule hits that reduce the increased mitochondrial stress (unfolded protein response, UPR^MT) that occurs in mitochondrial disease. Mitochondrial stress was quantified by analysis of the green to red normalized fluorescence ratio in a triple transgenic hsp-6p::gfp C. elegans N2 strain stably crossed to a myo-2p::mCherry reporter used to quantify animal number. Specifically, we developed a novel high-throughput methodology for inducing DLD RNAi knock-down using a triple transgenic hsp-6p::gfp C. elegans strain stably crossed to a myo-2p::mCherry reporter on solid media exposed in liquid RNAi media for 24-hour candidate drug exposures in a 384-well plate format.

Results: A HTS drug screen has been successfully developed and validated using positive control compounds previously identified in our research group to rescue UPR^MT using DLD deficient RNAi knockdown models of C. elegans in a solid to liquid RNAi media format (Broxton et al, 2022). Active screening of a 2,560 compound FDA-approved and natural product drug library (Spectrum Collection, MicroSource) is now underway in this model to identify candidate drugs hits that rescue UPR^MT back toward wild-type levels.

Conclusion: We have developed a HTS pipeline that is being used to interrogate effects on mitochondrial stress of a large FDA-approved drug and natural product library in a liquid feeding RNAi knockdown model of DLD deficiency in C. elegans. The goal of this work is to identify drug and natural product candidates that can potentially be rapidly repurposed to evaluate their safety and potential to improve health in human patients with DLD deficiency. In addition, the success of this HTS protocol in RNAi-induced nuclear gene knockdown models in C. elegans in a liquid 384-well plate format will enable HTS for therapeutic leads across a broad spectrum of primary mitochondrial diseases, including diseases where a knockout model is nonviable.

Abstract #: 2023PA-0000000183

Presenter: C. Pantano

Implementing the CHOP Mito Care Pathway to deliver clinical care in the inpatient setting

Pantano C^1*, Demczko M¹, Ganetzky R¹, Goldstein A¹

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA

*pantanoc@chop.edu

To standardize clinical care in a concise, readily accessible format for patients with primary mitochondrial disease (PMD) in the inpatient setting, physicians in the Mitochondrial Medicine Frontier Program (MMFP) at the Children’s Hospital of Philadelphia (CHOP) created the Mitochondrial Care Pathway (MCP). Launched in Spring 2022, the MCP has been utilized in multiple healthcare settings and clinical circumstances. Here, we describe three patient vignettes where utilization of the MCP streamlined medical management in a collaborative manner between mitochondrial disease experts and front-line clinicians.

Patient 1 is a 5-year-old girl with Leigh Syndrome caused by a pathogenic MT-TK variant, m.8344A>G at 97% heteroplasmy level in blood who presented to the emergency department with fever and congestion due to the SARS-CoV2 infection. Despite dehydration, appropriate fluid resuscitation was delayed due to provider uncertainty and concern that dextrose-containing fluid would induce lactic acidosis. The MCP provided recommendations and clarified misconceptions about fluid contraindications for patients with PMD. Intravenous fluids containing 5% dextrose were initiated at maintenance rate to prevent catabolism in the setting of illness.

Patient 2 is a 15-year-old female with a history of early childhood epilepsy, mild intellectual delay, and sensorineural hearing loss who was admitted to the pediatric intensive care unit for weight loss, significant leg cramping, encephalopathy, and lactic acidosis. In the setting of a possible (and ultimately genetically confirmed) PMD, recommendations for managing her lactic acidosis including specific buffering agent, dose and rate as determined by the MCP calculator. A sodium bicarbonate infusion was initiated and continued throughout the acute phase of her admission until lactate normalized and supplementation was eventually discontinued.

Patient 3 is a 3-month-old male with a history of failed hearing screen who presented to an outside hospital with new-onset seizures in the setting of rhinovirus, with subsequent brain MRI concerning for Leigh Syndrome. CHOP MMFP was consulted by the outside hospital and referred the primary team to the MCP for detailed clinical guidance. Following team collaboration per MCP recommendations, high-dose steroids were given, and intravenous arginine was held based upon MRI findings.

Care pathways are an invaluable tool to provide standardized high-quality care for complex patients at various points of health system contact. Until recently, care pathways were generally deemed inapplicable to the care of rare disease patients. While clinical judgement will always take precedence and the need for consultation with specialists remains inevitable, the MCP established by the CHOP MMFP team offers a publicly available tool to help clinicians and families navigate the challenges of an acute care situation in diverse health systems and points of contact. Overtime, MCP updates will continually standardize optimal management of acutely ill patients with PMD.

Abstract: 2023PA-0000000184

Presenter: Emily Bogush

Facilitating community-based genomic data analysis in primary mitochondrial disease: Mitochondrial Disease Sequence Data Resource (MSeqDR) and mitoSHARE patient registry collaboration to support genomic data discoveries

Bogush E^1*, McCormick EM¹, Qunell ES¹, Yeske PE², Gai X^3,4, Falk MJ^1,5

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, USA; ²United Mitochondrial Disease Foundation, USA; ³Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, USA; ⁴Keck School of Medicine, University of Southern California, USA; ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA

*bogushe@chop.edu

Primary mitochondrial diseases (PMD) are a highly heterogeneous group of disorders with pathogenic variants recognized in more than 300 genes across both nuclear and mitochondrial genomes. Much remains to be learned about PMD, as phenotypic variability exists among individuals with the same genetic disorder and many individuals with medical concerns highly concerning for PMD lack a confirmed genetic etiology. While clinical diagnostic genomic testing for PMD has become more accessible and widely utilized, analysis of these complex data often remains limited to clinical diagnostic laboratories that typically report only variants in genes previously associated with PMD. Opportunity exists for novel gene discovery or more complex genomic studies, such as pharmacogenomic or disease modifier analyses, of existing genomic data from PMD subjects.

Here, we describe a community-wide mechanism to facilitate research analysis of existing genomic data by the PMD global research community. Participants enrolled in the UMDF-led mitoSHARE mitochondrial disease patient registry who express interest in sharing their existing genomic raw data files can participate by completing a mitoSHARE survey to learn more about the Children’s Hospital of Philadelphia (CHOP) Institutional Review Board (IRB)-approved Mitochondrial Disease Sequence Data Resource (MSeqDR) genomic data repository. 27 registry participants who self-identified as eligible for this study contacted the CHOP research team (PI, MJF) to learn about the MSeqDR informed consent process, elected to proceed with study enrollment, and returned signed genomic data release forms to allow their existing raw genomic data to be obtained on their behalf from the testing laboratory by the study team. Return of signed genomic data release forms are pending for an additional 13 registry participants consented to the study.

Once obtained, coded genomic data is uploaded to a secure Cloud-based Web server, stripped of identifying information, and labeled with a global universal identifier (GUID) that is returned to the patient donor to share with specific researchers at their discretion to facilitate analyses of their own GUID-labeled genomic data within MSeqDR in a secure, Web-based central portal that enables genomics data analysis using sophisticated, user-friendly, data query platforms (e.g., OpenCGA, Genesis). Medical professionals with genomic data proficiency who are approved by the mitoSHARE-MSeqDR data use committee and provided by a patient or family with their unique GUID identifier will thereby be able to not just review printed reports but instead to readily analyze exome, genome, and/or mtDNA genome data from individuals and families with known or suspected PMD to attempt to find the underlying genetic etiology and potentially other research-based uses. No research results will be directly provided to families by the MSeqDR study team that is organizing this community-wide genomic data access and analysis effort, results may inform the approved researchers’ interrogation of disease etiology candidates for specific patients and/or family’s genomic data when specific GUIDs are shared directly by the family.

Overall, patient and family enrollment in the mitoSHARE-MSeqDR project continues the effort to improve success rates for genomic analyses of PMD.

Abstract #: 2023PA-0000000186

Presenter: Daniel McGinn

Racial Disparity in the Diagnosis of Mitochondrial Disease

McGinn DE¹, Muraresku CM², Valverde K¹, Falk MJ^2,3, Ganetzky R^2,3

¹Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; ²Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia PA, USA; ³Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA USA

daniel.mcginn@pennmedcine.upenn.edu

Primary Mitochondrial Diseases (PMD) are caused by inherited disruption of oxidative phosphorylation, the main process by which the body creates energy. Collectively, PMD are the most common inborn error of metabolism. PMD has extensive variability in symptoms, age at presentation, and severity. A 2020 North American study of individuals with PMD reported racial background in > 85% was white. This significantly differs from the 2020 US Census, in which 61% of Americans self-reported as white. Here, we report results of an initial investigation of racial disparities in ascertainment and disease severity assessment for PMD patients to determine if PMD patients of color may be under-diagnosed. Findings hypothesized to be suggestive of PMD under-diagnosis in patients of color would be higher disease severity scores in patients of color and increased chance of PMD diagnosis being made in the inpatient setting, as both would represent there being a higher symptomatic threshold required to consider and establish a PMD diagnosis in patients of color relative to those who self-identified as white.

We retrospectively reviewed electronic health records on 337 subjects with molecularly-confirmed PMD consented into the CHOP IRB-approved study (#08-6177). Probands were included in this study if sufficient data was available on demographics, diagnostic setting, and symptom severity (n=207). Symptom severity was assessed using a multisystem aggregate severity score index [SSI], where lower scores indicate more severe disease. For each patient, we also gathered information on possible confounding variables including socioeconomic status, proximity to CHOP, and insurance coverage.

The CHOP PMD cohort (n=207) was 53.6% female with a mean age of 18.1 years. In this cohort, patient-reported racial background was 74.9% white, 11.1% Hispanic, 9.7% Asian, and 7.2% Black. The average age at PMD diagnosis was 9.72 years, with 75.8% diagnosed in the outpatient setting. The average SSI was 13.55. Notably, Black (mean SSI=12.0, p=0.013) and Hispanic (mean SSI=12.5, p=0.016) patients were more likely to have more severe symptoms compared with PMD patients from white, non-Hispanic backgrounds (mean SSI=14.2). Notably, Asian, Black, and Hispanic PMD patients were substantially more likely to be diagnosed as inpatients (40%, 40%, and 35%, respectively) compared with white PMD patients (20%).

Self-identified PMD patients of color were more likely than white patients to be diagnosed with PMD in the inpatient setting. Additionally, Black and Hispanic patients tended to have more severe symptoms as compared with PMD patients from other backgrounds. These data support the premise that patients of color tend to meet a higher “diagnostic threshold” to achieve a diagnosis of PMD. Overall, these findings suggest that previously reported data showing a higher preponderance of PMD patients who are self-reported as white is likely influenced at least in part to ascertainment bias. People of color are likely under-diagnosed within the PMD community, representing a critical but previously unrecognized health disparity for people of color. As our data suggest that a major barrier to diagnosis is obtaining a referral to a mitochondrial medicine clinic, overcoming this disparity requires wide-scale education of referring physicians of pan-racial occurrence of PMD.

Abstract #: 2023PA-0000000187

Presenter: Graeme Preston

A Large-Scale Drug Screen For Compounds That Improve Reductive Stress In Melas Cardiomyocytes

Preston GP¹, Morava E¹ and Kozicz T¹

¹Department of Clinical Genomics, Mayo Clinic, USA

*Preston.Graeme@mayo.edu (corresponding author’s email)

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the most common mitochondrial disorders, affecting 1 in 4000 live births. It is most frequently caused by the m.3243A>G pathogenic variant in the mitochondrial transfer RNA gene MT-TL1. Cells of individuals with MELAS display a profound impairment of the mitochondrial electron transport chain, and reduced ATP synthesis. In addition to reduced cellular energy, these cells also display an increased abundance of NADH, and a concomitant depletion of NAD⁺, due to reduced flux through mitochondrial complex I. This increased ratio of NADH:NAD⁺, known as reductive stress, results in additional impairments of cell metabolism. Notably, individuals with MELAS also display increased rates of cardiomyopathy, characterized by concentric ventricular hypertension and ECG anomalies including long QRS complex and long QT syndrome, compared to patients with other mitochondrial diseases. We previously conducted a screen of ~2500 FDA-approved drug compounds on cardiomyocytes (iCMs) differentiated from induced pluripotent stem cells (iPSCs) derived from a single individual with 70% heteroplasmy of the m.3243A>G pathogenic variant. iCMs were virally transduced with a live fluorescent probe of the NADH:NAD⁺ ratio known as “SoNar”. We identified 23 compounds that significantly (SD>3) reduced the NADH:NAD⁺ ratio in these cells. Here, we report on a validation screen of these 23 compounds using iCMs to identify optimal drug repurposing candidates selected for highest potential to reverse cardiac issues with lowest risk of off-target toxicity in human cells. We differentiated iCMs from iPSCs derived from three individuals with 70% heteroplasmy of the m.3243A>G pathogenic variant and transduced them with the SoNar probe. iCMS were then exposed to 8 concentrations of the 23 FDA-approved compounds previously identified as potential mitigators of reductive stress in iCMs. The NADH:NAD⁺ ratio was measured at 2-, and 24-hours following exposure, and the shift in NADH:NAD⁺ ratio was calculated. High potential hits from this validation screen will inform optimized clinical trials to improve clinical care initially for individuals with m.3243A>G-related mitochondrial disease and then progress to individuals with other pathogenic variants associated with MELAS.

Abstract#: 2023PA-0000000191

Presenter: Fernando Scaglia

M.12315G>A Pathogenic Variant in MT-TL2 presenting with a MELAS-like Clinical Syndrome and Depletion of Nitric Oxide Donors

Snyder MT¹, Manor Y², Mizerik E¹, Gijavanekar C¹, Elsea SH¹, Machol K¹, Emrick L³, Scaglia F¹

¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas; ²Metabolic Diseases Unit, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Israel; ³Department of Pediatrics, Division of Pediatric Neurology and Developmental Neuroscience, Baylor College of Medicine, Houston, Texas

Problem: The mitochondrial tRNA leucine variant m.12315G>A in MT-TL2 has been previously reported in one patient with chronic progressive ophthalmoplegia, ptosis, pigmentary retinopathy, and sensorineural hearing loss and in a second patient with mitochondrial myopathy and ophthalmoplegia. Herein, we present the case of a patient harboring this pathogenic variant who developed clinical features of MELAS and exhibited depletion of arginine on plasma amino acid analysis and citrulline on untargeted metabolomics analysis. These findings in conjunction with the absence of nicotinamide and 1-methylnicotinamide on metabolomics analysis helped to redirect therapeutic approach in this patient.

Objectives: Describe a case of a rare mitochondrial DNA variant in MT-TL2 presenting with a MELAS-like phenotype whose biochemical studies helped to target a more specific therapeutic approach.

Methods: We report the case of a previously healthy two-year old child presenting with lethargy and severe lactic acidosis during a gastrointestinal illness.

Results: Brain MRI stroke protocol was normal, while brain MRS showed a lactate peak. Biochemical and genetic evaluations were performed. Plasma amino acids revealed elevations of alanine and low arginine. Trio whole exome sequencing was non-diagnostic. Next generation sequencing of the mitochondrial genome revealed a pathogenic variant in MT-TL2 (m.12315G>A) at 63% heteroplasmy in blood. GDF15 returned elevated at >6000 pg/mL (normal <750 pg/mL). Untargeted metabolomics analysis showed low citrulline (Z score -5.9) with absent nicotinamide and 1-methylnicotinamide. She was started on nicotinamide. At the time of discharge, she was NG-tube dependent, able to walk with guidance, and had difficulty with speech articulation. One year later she presented with focal right upper extremity weakness and seizures in the setting of a prolonged viral illness. MRI stroke protocol showed cytotoxic edema of the left temporal/occipital region which later progressed to right sided temporal/occipital cytotoxic edema with corresponding luxury perfusion. The presence of a metabolic stroke in a non-classic vascular distribution on MRI, and the review of historical plasma untargeted metabolomics data consistent with low citrulline prompted the initiation of intravenous arginine therapy. She was discharged on enteral citrulline and nicotinamide supplementation. Citrulline and nicotinamide levels on metabolomics normalized with therapy. Four months later she developed right-sided ptosis and seizures in the setting of illness. MRI at that time showed acute cytotoxic edema in the bilateral posterior hippocampi and right dorsomedial thalamus. Intravenous arginine was given with improvement in her neurological symptoms.

Conclusion: The mitochondrial variant m.12315G>A has been reported previously in two other individuals with milder phenotypes in the setting of lower heteroplasmy levels in blood. Our patient is more severely affected since she presents with clinical features of MELAS. Her metabolic strokes may be due to decreased nitric oxide availability indicated by low plasma citrulline on untargeted metabolomics analysis and low plasma arginine on plasma amino acids. The use of intravenous arginine during acute stroke-like episodes may provide significant benefit for patients with this m.12315G>A variant and associated NO depletion. These findings expand the clinical spectrum associated with this pathogenic variant.

Abstract: 2023PA-0000000192

Presenter: Suraiya Haroon

Identification of two mitophagy modulators that rescue mitochondrial stress in a heteroplasmic single large-scale mtDNA deletion (SLSMD) C. elegans animal model

Mendel R¹, Cheng Y¹, Tara Z¹, Schrope S¹, Keith K³, Mathew N¹, Ogiso E¹, Falk MJ^1,2, and Haroon S^1,2*

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA; ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; ³Department of Biomedical and Health Informatics (DBHi), Children’s Hospital of Philadelphia, Philadelphia, PA

*haroons@chop.edu

Kearns-Sayre syndrome (KSS) and Pearson Syndrome (PS) are primary mitochondrial diseases caused by single large-scale mitochondrial DNA (mtDNA) deletions (SLSMD). SLSMD diseases, as with most mitochondrial diseases, have no effective treatments or FDA approved therapies. One major roadblock to therapeutic development is the lack of tools to genetically engineer models harboring mtDNA deletions that are stable and transgenerational. As such, studying naturally occurring animal models help to advance understanding of disease progression and enable therapeutic development. Specifically, the uaDf5 C. elegans model harbors a heteroplasmic 3.1 kilobase mtDNA deletion of 11 genes that encode 7 MT-tRNAs and 4 mitochondrial proteins. Here, we use a mitochondrial stress phenotype in the uaDf5 animals to screen for potential therapeutic leads from two libraries consisting of 62 mitophagy modulators and 2,560 FDA-approved drugs and natural products. To conduct the screen, uaDf5 C. elegans were crossed with a myo2::mcherry reporter, a red fluorescent pharyngeal bulb marker for automated animal count, and an hsp6p::GFP reporter that fluoresces green upon mitochondrial stress induction. These triple transgenic animals were used to screen potential therapies using a CX5 high content imager (ThermoFisher). 3 drugs tested from empirically identified compounds previously found to rescue survival in a C. elegans gas-1(fc21) complex I disease model were identified as positive controls for the library screen based on their ability to rescue mitochondrial stress in uaDf5 animals. Upon screening the mitophagy modulator library, 5 additional compounds were identified that significantly rescued mitochondrial stress in uaDf5 mutant animals. Testing these 5 compounds at 4 different concentrations (12.5 µM to 100 µM) revealed only 2 that reproducibly rescued mitochondrial stress in a dose-dependent manner across multiple biological replicates. Studies to validate the ability of these two modulators to rescue SLSMD heteroplasmy levels in uaDf5 worms is underway, as well as combination treatments of control compounds and mitophagy modulators. Further, a high-throughput screen of a 2,560 FDA-approved drug and natural product library identified 69 hits now being retested. Once compounds pass the retest in the C. elegans model, leads will be tested in 6 human SLSMD patient fibroblast cell lines exposed to metabolic stress conditions were used to evaluate lead therapy effects on cell survival (ATP levels), SLSMD heteroplasmy levels (qPCR), mitochondrial content (MitoTracker Green fluorescence, western immunoblot analysis), and integrated mitochondrial respiration (Seahorse). In conclusion, the uaDf5 heteroplasmic SLSMD worms enable mechanistic and therapeutic modeling insights, as well as high throughput screening of drug libraries to identify novel therapeutic leads for SLSMD diseases. Two mitophagy modulators and three known mitochondrial disease modulating compounds have been identified to rescue the mitochondrial stress phenotype in the SLSMD C. elegans model, with additional potential leads now under validation that hold potential for future clinical research studies to treat human SLSMD diseases.

Abstract #: 2023PA-0000000193

Presenter: Mithal Divakar

Cardiac Manifestations Of Mitochondrial Disease: A Single Center Experience

Lottes, R.G. MD, PhD^1,3 andMithal, D.S. MD, PhD^2,3*

¹Section of Cardiology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, 60611; ²Section of Neurology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, 60611; ³Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611

*divakar@northwestern.edu

Primary mitochondrial diseases (PMDs) are known to effect the heart but often go unrecognized due to inconsistent screening protocols and a paucity of primary data on cardiac manifestations. A recent, growing body of evidence indicates that cardiovascular disease is a major cause of morbidity and mortality in children with PMDs. This study aims to describe the phenotypes and frequency of cardiovascular disease in a single-center cohort. The study is a single center, retrospective cohort of 86 children with mitochondrial disease was interrogated for evidence of structural and/or electrophysiological cardiac disease. Charts were reviewed and clinical and genetic characteristics were extracted. Demographic, echocardiographic, and electrocardiographic data was recorded. Instances of orthotopic heart transplant and outcomes is also specifically reported. In the results we report a wide variety of structural and electrophysiologic disease was identified in this patient cohort. The features for both echocardiography (e.g. ejection fraction) and electrocardiography (e.g. PR interval) are reported in table format. 25/86 (29%) of patients had definitive diagnoses or highly suggestive findings of cardiac disease. Some patients had multiple cardiac diagnoses, with 30 total diagnoses assigned to 86 patients. Three patients had heart transplant, and details are provided in a separate table. The most common structural diagnoses were hypertrophic cardiomyopathy and dilated cardiomyopathy. The most common electrophysiologic diagnosis was Wolf-Parkinson-White Syndrome. Of the 25 patients with known cardiac disease, mortality rate was apparently higher (10/25, 40%) versus those without (17/61, 28%), although the difference failed to meet significance (p=0.19, Pearson correlation). We conclude that patients with mitochondrial disease in this single-center study have a significant burden of cardiovascular disease including both structural and electrophysiologic manifestations. The mortality risk of patients with PMD and cardiovascular manifestations is high, but not significantly different than those without known heart disease. Further studies are required to understand which patients are at highest risk of cardiovascular complications and how best to screen and manage these patients going forward.

Abstract #: 2023PA-0000000194

Presenter: Leonard Burg

Modeling Exercise Intolerance Through Maximal Swimming Capacity And Whole Body Oxygen Consumption Capacity With Exercise In Adult Zebrafish Models Of Mitochondrial Disease

Burg L¹, Reesey Gretzmacher E¹, Falk M.J.^1,2

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; ²Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

BurgL@CHOP.edu

Mitochondrial respiratory chain defects caused by pathogenic mutations in both nuclear and mitochondrial DNA genes can result in impaired exercise capacity and reduced ability of muscles to utilize oxygen. Patients with mitochondrial respiratory chain defects may exhibit exercise intolerance, where limited amounts of physical exertion may induce muscle fatigue due to an inability to utilize the increased amounts of oxygen delivered to muscle during physical exercise. Zebrafish (Danio rerio) have gained increased utility in recent years to model the basic mechanisms of diverse human diseases due to a variety of forward and reverse genetic approaches and their relative ease of use for large-scale pharmacologic or genetic library screens. Here, we tested several zebrafish animal models of mitochondrial disease for exercise intolerance by measuring their maximal swimming capacity and oxygen consumption rates during exercise. Exercise intolerance in surf1^-/- (complex IV deficiency), fbxl4^-/- (multiple respiratory chain complex deficiency), and wild-type control adult zebrafish older than 3 months post fertilization were exercised in a Loligo swim tunnel equipped with a respirometer to simultaneously measure maximal swimming capacity and oxygen consumption rates (M0₂) as fish swam against an incrementally increased current until exhaustion was reached. Candidate therapies will be tested for their ability to rescue impairment in mutants’ maximal swimming capacity and oxygen consumption rates. In adult surf1^-/- zebrafish, we observed a significantly decreased capacity for maximal swimming speed compared to age-matched wild-type siblings performing the same exercise protocol. Additionally, adult surf1^-/- zebrafish were found to have significantly decreased M0₂ during vigorous exercise when compared to wild-type siblings. In contrast, adult fbxl4^-/- zebrafish were found to have no significant reduction in either maximal swimming capacity or M0₂ as compared to their age-matched wild-type siblings siblings performing the same swimming exercise protocol. Zebrafish animal models of primary mitochondrial diseases enable quantitation of their whole body exercise capacity. Interestingly, while surf1^-/- animals displayed significantly reduced maximal swimming capacity and M0₂, fbxl4^-/- adult zebrafish demonstrated normal exercise capacity. Exercise tolerance testing of other primary mitochondrial disease zebrafish models created by our research laboratory underway, combined with testing the effects of candidate therapeutic leads. These studies will provide further insight towards development of translational therapies for primary mitochondrial disease patients with exercise intolerance.

Abstract #: 2023PA-0000000195

Presenter: Ngoc Hoang

Sex-specific colonic mitochondrial dysfunction and improvement with mitochondrial targeted therapies in the indomethacin-induced inflammatory bowel disease model in rats

Hoang*, N., Brooks, K., and Edwards, K.

Department of Cell and Molecular Biology, University of Mississippi Medical Center Jackson, MS

*Corresponding author: nhhoang@umc.edu

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract and encompasses Crohn’s disease and ulcerative colitis. Women appear to have more severe and recurring symptoms of IBD compared to men, most likely due to hormonal fluctuations. Many studies have shown that mitochondrial dysfunction plays a role in the development of inflammation and that colon mitochondrial alterations are observed in IBD patients. In this study we have identified the presence of sex-specific colon mitochondrial dysfunction in a rat model of IBD. Eight-week-old male and female rats received two doses of indomethacin (7.5 mg/kg) 24 hour apart to induce IBD. MitoTEMPO (1mg/kg/day) groups received injections either following indomethacin injection or before indomethacin injections until the end of the experiment. Colon tissues were collected for mitochondrial isolation two days after injections. Mitochondrial respiration and reactive oxygen species (mtROS) production were measured simultaneously by an Oroboros Fluorespirometer using glutamate/malate, succinate, oleate (long-chain fatty acid; LCFA), or octanoate (medium chain fatty acid; MCFA). The activities of the individual electron transport complexes antioxidant enzymes were also measured to assess mitochondrial function. Data were normalized to mitochondrial content using citrate synthase (CS) activity, which confirms that the observed changes are not due to loss of mitochondrial content. At days 2 and 3 following injection, IBD rats showed a significant (p<0.05) decrease in body weight, food intact, and an increase colon inflammation indicating the peak of the disease. Therefore, animals were used on day 2 or 3 following injections. IBD male rats showed a significant (p<0.05) decrease in citrate synthase activity (38%), cardiolipin levels (48%), catalase activity (37%), and an increase in mtROS production (193%). IBD females show a significant (p<0.05) decrease in intact mitochondrial respiration (45%), RCR (57%), complex I (57%) and IV (31%) activity, and an increase in mtROS (265%). Interestingly, control females showed a significantly (p<0.05) higher rate of complex I (152%) and II-driven (158%) intact mitochondrial respiration, MCFA oxidation (126%), and complex II (153%), III (160%), and IV (135%) activities compared to control males. The use of a mitochondrial-targeted therapy, mitoTEMPO, significantly (p<0.05) improves the disease activity, decreases mtROS, and colon mitochondrial function in in female IBD rats. However, in the males there was no observed improvement, likely due to the decrease in catalase activity. Our study provides a better understanding of the role mitochondria in the development of IBD and highlights sex differences in mitochondrial function. It also opens an avenue for the development of strategies to re-establish normal mitochondrial function that could provide more options for preventive and therapeutic interventions for IBD.

Abstract #: 2023PA-0000000198

Presenter: Chenxu Li/Marcia Terluk

Targeting Mitochondrial Metabolism using Nervonic Acid in Adrenoleukodystrophy

Terluk M.R., Li C., Kartha R.V.* Chenxu Li

Center for Orphan Drug Research, Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN, USA

*rvkartha@umn.edu (Corresponding author’s email)

Adrenoleukodystrophy (ALD) is an X-linked neurodegenerative disorder caused by a mutation in the ABCD1 gene, which results in metabolic abnormalities of very long-chain fatty acids (VLCFA) and VLCFA-CoA esters, disturbing their proper degradation within the peroxisome. Elevated levels of VLCFA would then trigger adrenal insufficiency and progressive demyelination in the central and peripheral nervous systems of patients with ALD. The clinical manifestations of ALD display a wide range of variations, and there is no known genotype-phenotype correlation. The two common phenotypes of ALD are adrenomyeloneuropathy (AMN), mainly affecting adult males and heterozygous women, and cerebral ALD, which primarily affects boys. Mitochondrial dysfunction has been reported in patients, mouse, and cell models of ALD, indicating the contribution of abnormal mitochondria as an underlying pathological mechanism to peroxisomal disorders. We have shown that nervonic acid (NA, C24:1), a monounsaturated dietary fatty acid, protects ALD fibroblasts from H₂O₂-induced oxidative stress and increases ATP production. In the current study, we aim to investigate further the effect of NA on the mitochondrial metabolic pathways in skin fibroblasts derived from AMN patient (GM17819). Cells were treated with NA (20 µM) and analyzed using the Substrate Oxidation Stress Test with a low glucose medium and XFe96 Extracellular Flux Analyzer (Agilent Technologies). We analyzed the effect of NA on glucose/pyruvate (Glu), glutamine (Gln), and long-chain fatty acid (LCFA) pathways using specific inhibitors UK5099, BPTES, and etomoxir, respectively. The cells also were sequentially exposed to additional modulators, including oligomycin, trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and a combination of rotenone and antimycin A to assess the basal and maximal mitochondrial respiration profile. The extracellular oxygen consumption rate (OCR) results were analyzed using the Seahorse Wave Pro and Seahorse Analytics software from Agilent. After 24 hours of treatment with 20 µM NA, we observed a significant increase in OCR for basal and maximal respiration for all three pathways (Glu, Gln, and LCFA). This increase was observed to be 2.1-fold for both parameters (p<0.05, n=4). In addition, in the presence of inhibitors to block each of these pathways, NA treatment showed an increasing trend in basal and maximal respiration in the groups treated with either BPTES or etomoxir alone or combined with BPTES and UK5099. Our findings show that NA improves all three primary mitochondrial metabolic pathways in AMN cells, especially the Gln and LCFA-dependent pathways in low glucose conditions. In general, NA contributes to improving mitochondrial function and has the potential to be used for treating patients with ALD and, by extension, other neurological diseases with associated mitochondrial dysfunction.

Abstract #: 2023PA-0000000200

Presenter: Irene Yee

Succinate Does Not Increase Reactive Oxygen Species Generation In Phosphorylating Human Mitochondria

Yee I.^1*, Lenzer A.¹, Sekine S.^1,2, Liu T.¹, Chamkha I.^1,3, Elmér E.^1,3, Ehinger J. K.^1,4

1. Mitochondrial Medicine, Department of Clinical Sciences, Lund University, Sweden; 2. Department of Anesthesiology, Tokyo Medical University, Japan; 3. Abliva AB, Sweden; 4. Otorhinolaryngology Head and Neck Surgery, Department of Clinical Sciences, Lund University, Skåne University Hospital, Sweden

*irene.yee@med.lu.se

Reactive oxygen species (ROS) are generated in metabolism and play a role in cellular signalling as well as in a wide range of human diseases. However, it has proven a formidable challenge to detect and interpret the physiological and pathophysiological role of mitochondrial and cellular ROS production under experimental conditions. One major site of ROS production is thought to be complex I (CI) of the mitochondrial respiratory system through reverse electron transport (RET), during which electrons flow from complex II (CII) back through ubiquinol to CI. It has been proposed that RET can occur during cellular succinate accumulation and metabolism, as in ischemia with reperfusion. However, most experimental studies detecting increased ROS production by succinate have been carried out with downstream (of CII) block of electron transport or mitochondrial ATP synthesis. The aim of our study was to re-evaluate the effects of succinate metabolism on ROS generation in phosphorylating mitochondria, using intact and permeabilized freshly isolated human peripheral blood cells (PBMCs), as well as crudely isolated human mitochondria. Mitochondrial membrane potential (TMRM), mitochondrial ROS levels (mitoSOX), and cytoplasmic ROS (DHE) levels were visualized using flow cytometry. Mitochondrial respiration and hydrogen peroxide levels (Amplex UltraRed assay) were assessed with the Oroboros O2k FluoRespirometer. For intact PBMCs, succinate was delivered intracellularly using two different prodrug strategies (NV189, NV354). Both non-inhibited and CI-inhibited intact cells displayed increased respiration and membrane potential, while showing a decreased release of hydrogen peroxide and production of ROS. In permeabilized non-inhibited and CI-inhibited PBMCs, as well as crudely isolated mitochondria from PBMCs, addition of native succinate did not increase hydrogen peroxide production in the phosphorylating state. We conclude that succinate does not increase ROS generation in phosphorylating human mitochondria. Further, intracellular delivery of succinate to intact cells decreases production of mitochondrial and cytoplasmic ROS as well as the cellular release of hydrogen peroxide.

Abstract #: 2023PA-0000000201

Presenter: Nicole A. Woodard

Development Of Osteosarcoma Xenografts In Zebrafish Models Of Mitochondrial Disease

Woodard NA^1*, Dalwadi S¹, Patterson L², Nakamaru-Ogiso E^1,3, Resnick AC^2,4 and Falk MJ^1,3

¹Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, USA; ²Center for Data Driven Discovery in Biomedicine, Department of Surgery, The Children’s Hospital of Philadelphia, USA; ³Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA; ⁴Department of Neurosurgery, The Children’s Hospital of Philadelphia, USA

*woodardn@chop.edu

While primary mitochondrial diseases (PMD) and osteosarcoma are both developmental diseases, it is remarkably rare to find a clinical presentation of cancer in patients with PMD. Both diseases favor the less energy efficient but carbon-sparing glycolytic pathway over the respiratory chain for ATP generation, known as the “Warburg effect”. To understand further connections between PMD and cancer, we have developed osteosarcoma xenografts in established zebrafish models of mitochondrial disease. Osteosarcoma cell lines 143B, MG-63, and U-2 OS were transduced via lentivirus to express dual reporters: green fluorescent protein (GFP) and luciferase. The dual-labeled osteosarcoma cells were harvested and transplanted into 2-day post-fertilization wild-type, NDUFS2^-/- (complex I deficient), and SURF1^-/- (complex IV deficient) zebrafish at the duct of Cuvier and monitored for cell migration, tumor growth and proliferation, tumor rejection, and metastasis. The NDUFS2^-/- zebrafish is an embryonic-lethal model that was monitored for the length of its lifespan through ~9-days post-fertilization, while wild-type and SURF1^-/- zebrafish were followed for 6 weeks. Having established these novel zebrafish xenograft models, future studies will apply multi-omics and bioinformatics analyses to compare the bioenergetics, metabolic, signaling, and immune pathways that may differ between osteosarcoma and PMD in an effort to discover new therapeutic targets and/or candidate treatment options for both classes of developmental disease.

Abstract #: 2023PA-0000000202

Presenter: John Campbell

Biomarker Cardiolipin Ratios Predict Phenotypic Responders in Barth Syndrome Patients with Cardiomyopathy: Analysis from TAZPOWER OLE 168 Week Study

Vernon HJ¹, Hornby BD²; Thompson WR³; *Campbell J⁴; Abbruscato A⁴; Carr J⁴; Brown D⁴

¹McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine and at the Kennedy Krieger Institute; Director, Barth Syndrome Clinic at Kennedy Krieger Institute, Baltimore, MD, USA; ²Department of Physical Therapy, Kennedy Krieger Institute, Baltimore, MD, USA; ³Division of Pediatric Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD USA; ⁴Stealth BioTherapeutics, Needham, MA, USA

*John.Campbell@stealthbt.com

Barth syndrome (BTHS) is a rare, X-linked infantile-onset disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delays and neutropenia. BTHS is caused by defects in the TAFAZZIN gene that encodes tafazzin, a transacylase responsible for the remodeling and maturation of the mitochondrial phospholipid cardiolipin (CL). CL is critical to mitochondrial structure and function. In BTHS, the ratio of structurally immature monolyso-cardiolipin (MLCL) to structurally mature tetralinoleyl-cardiolipin (L4-CL) is significantly increased (MLCL:L4-CL ratio) above normal values. Abnormalities in the MLCL/CL ratio has 100% diagnostic specificity and sensitivity for BTHS and within the range of MLCL/CL ratios identified in patients, a subset of patients with quantitatively milder abnormalities and a relatively milder phenotype has been identified. In preclinical studies, elamipretide stabilized L4-CL, reduced oxidative stress, improved ATP generation, and increased TAZ gene expression. In the Phase 2/3 clinical study, TAZPOWER¹, a reduction in the MLCL:L4-CL ratio correlated with improved functional assessments, reduced incidence of neutropenia, and improved left ventricular volumes. Here we present the effect of elamipretide on MLCL:L4-CL ratios in BTHS patients who continued therapy in the Open-Label Extension (OLE) portion of the TAZPOWER study, which lasted 168 weeks. Efficacy in the OLE was measured by evaluating echocardiography, functional assessments (6-Minute Walk Test [6MWT], muscle strength via handheld dynamometry [HHD], 5-times-sit-to-stand [5XSST], and SWAY Balance score), patient-/clinician-/caregiver-reported questionnaires (including the BarTH Syndrome Symptom Assessment [BTHS-SA], PROMIS Short Form Fatigue, and Global Impression scales), and biomarkers, including MLCL:L4-CL ratios. Ten patients entered the OLE; 8 reached the Week 168 visit. Significant improvements from baseline on the 6MWT were seen at all OLE timepoints (cumulative mean 96.1 meters of improvement [Week 168, p=0.003]). Elamipretide treatment improved mean 6MWT performance and patient-/clinician-/caregiver-reported outcomes from baseline at all OLE timepoints. In addition, 3-D left ventricular stroke, end-diastolic, and end-systolic volumes, and the incidence of neutropenia, trended towards significant improvements from baseline to Week 168. Interestingly, using the mass spectrometric assay in blood spot (UMC, Amsterdam) the ratio of the remodeling intermediate monolysocardiolipin (MLCL) to mature cardiolipin (CL) species improved significantly in OLE completers (n=8) consistent with improvements observed in both the functional and echocardiographic assessments. The mean change in MLCL:CL ratios from baseline at week 168 for the 72:8 and 18:2 species was -86.15% (8.13 to 1.13) and -45.81 % (20.41 to 11.06) respectively. Considering the improvements observed in functional, echocardiographic and patient-reported outcome assessments, the correlating changes in the MLCL:CL ratios may suggest a molecular basis for the efficacy improvements observed with long-term elamipretide therapy in the TAZPOWER OLE. The mass spectrometric assay in blood spot is generally not considered to be reliably quantitative however the magnitude of change and the directional correlation with the functional and echocardiographic improvements is intriguing and warrants further study. Daily elamipretide was generally well tolerated, with injection site reactions being the most common adverse events.

Abstract #: 2023PA-0000000203

Presenter: Mikhail Alexeyev

TFAM C-terminal Tail is Dispensable for mtDNA Transcription and Replication

Kozhukhar N¹. and Alexeyev M.F^1*

¹University of South Alabama, Department of Physiology and Cell Biology

*malexeye@southalabama.edu

Mitochondrial transcription factor A (TFAM) is one of the best-studied but still incompletely understood mitochondrial protein, which plays a crucial role in the maintenance and transcription of mitochondrial DNA (mtDNA). The available experimental evidence is often contradictory in assigning the same function to various TFAM domains, partly owing to the limitations of available experimental systems. Recently, we developed the GeneSwap approach, which enables in situ reverse genetic analysis of mtDNA replication and transcription and is devoid of many of the limitations of the previously used techniques. Here, we utilized this approach to analyze the contributions of the TFAM C-terminal (tail) domain to mtDNA transcription and replication. We determined, at a single amino acid (aa) resolution, the TFAM tail requirements for in situ mtDNA replication in murine cells and established that tail-less TFAM supports both mtDNA replication and transcription. Unexpectedly, in cells expressing either C-terminally truncated murine TFAM or DNA-bending human TFAM mutant L6, HSP1 transcription was impaired to a greater extent than LSP transcription. Our findings are incompatible with the prevailing model of mtDNA transcription and suggest the need for further refinement.

Abstract #: 2023PA-0000000204

Presenter: Mikhail Alexeyev

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

Kozhukhar N¹., Spadafora D¹., Rodriguez Y.A.R¹. and Alexeyev M.F^1*

¹University of South Alabama, Department of Physiology and Cell Biology, USA

*malexeye@southalabama.edu

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

Abstract #: 2023PA-0000000205

Presenter: Mikhail Alexeyev

TFAM's Contributions to mtDNA Replication and OXPHOS Biogenesis are Genetically Separable

Kozhukhar N¹. and Alexeyev M.F^1*

¹University of South Alabama, Department of Physiology and Cell Biology, USA

*malexeye@southalabama.edu

The ability of animal orthologs of human mitochondrial transcription factor A (hTFAM) to support replication of human mitochondrial DNA (hmtDNA) does not follow a simple pattern of phylogenetic closeness or sequence similarity. In particular, TFAM from chicken (Gallus gallus, chTFAM), unlike TFAM from the “living fossil” fish coelacanth (Latimeria chalumnae), cannot support hmtDNA replication. Here, we implemented the recently developed GeneSwap approach for reverse genetic analysis of chTFAM to obtain insights into this apparent contradiction. By implementing limited “humanization” of chTFAM focused either on amino acid residues that make DNA contacts, or the ones with significant variances in side chains, we isolated two variants, Ch13 and Ch22. The former has a low mtDNA copy number (mtCN) but robust respiration. The converse is true of Ch22. Ch13 and Ch22 complement each other's deficiencies. Opposite directionalities of changes in mtCN and respiration were also observed in cells expressing frog TFAM. This led us to conclude that TFAM's contributions to mtDNA replication and respiratory chain biogenesis are genetically separable. We also present evidence that TFAM residues that make DNA contacts play the leading role in mtDNA replication. Finally, we present evidence for a novel mode of regulation of the respiratory chain biogenesis by regulating the supply of rRNA subunits.

Abstract #: 2023PA-0000000206

Presenter: Amy Goldstein

Single Large-Scale Mitochondrial DNA Deletion Syndromes (SLSMDS) Overlap with Rothmund-Thomson Syndrome (RTS): Two New Cases and a Review of the Literature

Freeman J¹, Elsharkawi I², Peterson J³, Kress D⁴, Ganetzky R^3,5, Falk MJ^{3, 5}, Goldstein AC^3,5

¹Pediatric Residency Program, Department of Pediatrics, University of Maryland Medical Center, Baltimore, Maryland; ²Medical Biochemical Genetics Fellowship, Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts ³Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ⁴Department of Dermatology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; ⁵Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania

Rothmund-Thomson Syndrome (RTS) is a dermatologic condition characterized by an early-onset rash consistent with poikiloderma, sparse hair, short stature, juvenile cataracts, skeletal and dental abnormalities, predisposition to certain cancers, and hematologic abnormalities including bone marrow involvement. In addition, symptoms may include gastrointestinal dysfunction, feeding intolerance, and failure to thrive. RTS is caused by biallelic pathologic intronic variants in RECQL4 or ANAPC1. The differential diagnosis for RTS in patients with negative genetic testing for these two known etiologies should include single large-scale mitochondrial DNA deletion syndromes (SLSMDs), which are clinically classified as discrete clinical phenotypes of Pearson Syndrome (PS), Kearns-Sayre Syndrome (KSS), and Chronic Progressive External Ophthalmoplegia (CPEO). Significant phenotypic overlap exists between RTS and SLSMDs, and skin findings can be one of the earliest manifestations preceding the classic features of PS that include transfusion-dependent anemia and exocrine pancreatic dysfunction.

Here, we present two cases followed in the Children’s Hospital of Philadelphia Mitochondrial Medicine Frontier Program clinic, one of whom had been clinically diagnosed with RTS until further manifestations of SLSMDs appeared. Literature review for previously reported cases of SLSMDs with dermatologic phenomena characteristic of RTS identified 13 additional cases. Collectively, this clinical case cohort reinforces the underreporting of skin manifestations in children with SLSMDs, with no mention of skin manifestations in many larger and more recent studies of SLSMD patients.

Overall, these cases provide evidence to include SLSMDs in the differential diagnosis of RTS in individuals who are not found to harbor biallelic pathologic intronic variants in RECQL4 or ANAPC1. In addition, these cases demonstrate the extensive overlap of clinical features of RTS and SLSMDs. We strongly recommend consideration for clinical mitochondrial genome sequencing and deletion studies in the genetic diagnostic evaluation of RTS spectrum patients.

Abstract #: 2023PA-0000000207

Presenter: Andrea Hoffmeier

Navigating the Mysteries of mtDNA Disease: Collaborative Patient-Specialist Communication in Clinical Trials

Hoffmeier¹, A., Chiaramello², A., Gropman, A.³

¹SHERPA Institute, Principal Investigator, MLA Harvard University, USA; ²Founding Director - Mito-EpiGen Program, George Washington University, School of Medicine & Health Sciences, USA; ³Neurogeneticist, Children's National Medical Center, USA

andrea@sherpainstitute.org

Primarily through the lens of patients, with insights from their research/medical care partners, this paper and presentation detail the importance of collaborative communication. The mysteries of mtDNA Disease are aggravating factors for many patients; it can take months or years for many patients to get a valid diagnosis. This period of ambiguity can cause substantial stress, with the potential to aggravate symptoms. Once a diagnosis is reached, it is common for patients to continue struggling to form productive relationships with researchers and medical professionals. Clinical trials can be especially unnerving for patients and their loved ones.

Too often, patients receive watered-down explanations about Mito-systems and how a disease manifests. A challenge for Mito Professionals is understanding the fit for each patient, in how deep to dive into details. This paper documents examples of Mito Professionals collaboratively communicating with patients, and “meeting them where they are” in understanding technical information. The inspiration for this study comes from real world patient-specialist conversations, and the level of technical detail that the latter is typically reticent to unpack for the lay person. Savvy specialists use metaphors and imagery to translate complex concepts into relatable information. Having a better appreciation for how the disease works, and the biological systems involved, helps tremendously in mitigating patients’ and their families’ stress.

A survey of patients (by a patient/social scientist) followed by interviews, will reveal further insights into this topic. This should benefit specialists in their patient interactions, as well as patients themselves, in preparing for their clinic appointments. As an example, it is not uncommon for Mitochondrial patients to have proprioceptive issues; however, they are often only cognizant of numbness causing balance problems. Gaining an awareness of something like proprioceptive issues, versus basic numbness, can make all the difference in coping with the disease and improving quality of life.

Knowledge is not a panacea (and certainly not a cure), but it can be a great comfort on the Mito Disease journey. Patients can make better decisions about activities and adjust expectations for what they can realistically do, or not do. Critical knowledge that makes all the difference starts with a patient-specialist collaboration and this presentation aims to offer novel insights into cultivating this vital relationship.

Abstract #: 2023PA-0000000208

Presenter: Gulam Syed

Dengue Virus Triggers Inflammation by Disrupting Host Mitochondrial Quality Control and Homeostasis

Bharati Singh^1*, Kiran Avula^1*, Shamim Akhtar Sufi^1*, and Gulam Hussain Syed^1#

DBT-Institute of Life Sciences, Bhubaneswar

*Equal Contribution, # Corresponding author

Mitochondrial fitness is governed by quality control system comprising mitochondrial dynamics and mitochondria-selective autophagy (mitophagy). Disruption of these processes is implicated in many human diseases including viral infections. We comprehensively analyzed the dengue virus's effect on multiple aspects of host mitochondrial homeostasis and its link to dengue disease pathogenesis. Despite a severe mitochondrial injury in infected cells, we observe that mitochondrial quality control and biogenesis are attenuated in dengue-infected human liver cells. This lead to the disruption of mitochondrial homeostasis and the onset of cellular injury and necrotic death in the infected cells. Paradoxically dengue promotes global autophagy but selectively disrupts mitophagy. Dengue virus disrupts the identification of the damaged mitochondria for elimination through mitophagy by downregulating the expression of PINK1 and Parkin, the two major mitophagy proteins. Mitophagy flux assays also suggest that Parkin-independent pathways of mitophagy are also inactive during dengue infection. Dengue infection also leads to the inhibition of mitochondrial biogenesis associated with a decline in the expression of the master regulators of mitochondrial biogenesis, PPARγ, and PGC1α. We observed that Dengue-infected cells release mitochondrial-damage-associated molecular patterns (mtDAMPs) such as mitochondrial DNA into the cytosol and extracellular milieu that can trigger naïve immune cells and promote pro-inflammatory signaling. Further investigation revealed that mtDNA release from dengue-infected cells was both dependent and independent of exosomes. The challenge of naive immune cells with culture supernatants from dengue-infected liver cells triggers the activation of NLRP3 inflammasome, pyroptosis, and proinflammatory signaling. In correlation to our in vitro observations, we observed that dengue patients have high levels of cell-free mitochondrial DNA in the blood acute phase of infection in comparison to the convalescent phase. We also observed that the level of cell-free mtDNA in blood strongly correlated with the degree of thrombocytopenia. Overall, our study shows how defective mitochondrial homeostasis in dengue-infected liver cells can trigger systemic inflammation and promote dengue disease immunopathogenesis.

Abstract #: 2023PA-0000000209

Presenter: Mai Sirimanne

Clinical Manifestations and Disease Burden of Primary Mitochondrial Myopathies (PMM): Results from a Patient Journey Analysis Shows Substantial Healthcare Resource Utilization

Sirimanne M^1*, Kates J¹, Saikumar S², Warner M³, Shah S², Lovink A², Yuqing X²

¹Reneo Pharmaceuticals, Inc., Irvine, California, USA; ²Trinity Life Sciences LLC, Waltham, Massachusetts, USA; ³Commercial Rx, Inc., Corona del Mar, California, USA

*msirimanne@reneopharma.com

Primary mitochondrial myopathies (PMM) are a group of disabling and underdiagnosed rare genetic disorders characterized by a range of clinical presentations and multisystemic impact. Diagnosis and management of PMM can be challenging due to the heterogeneity of clinical manifestations, including age of onset. There are no approved treatments for PMM. Moreover, current symptom management is not optimal in treating the underlying disease or enabling patients to improve their physical and social functioning. A patient journey analysis was performed to quantify the barriers that US patients face in their odyssey from clinical manifestation to diagnosis to symptom management. A cohort was extracted from Komodo closed-claims data for US patients who had a claim between 2016-2021. Due to the absence of a PMM specific ICD-10 diagnosis code, a stepwise approach was needed to identify patients with a suspected PMM. This entailed using ICD-10 diagnosis codes that were specific to mitochondrial disorders (MD), including chronic progressive external ophthalmoplegia, Kearns-Sayre Syndrome, and Leigh Syndrome. Analyses were then limited to patients who had ⩾1 myopathy claim, which represented >70% of patients in the initial sample. Patients who had a secondary MD ICD-10 code were excluded. Of 3.7K patients who were included for analysis, 97% experienced multi-organ manifestation, with impact across an average of 6 different organ systems (e.g., nervous, cardiac, musculoskeletal). Notably, patients experienced an increase in reported manifestations in the year prior to their diagnosis, with 73% patients reporting nervous system manifestations and ~70% patients reporting skeletal/muscular manifestation in the 12 months prior to diagnosis as compared to ~57% and 56% in the 24-36 months prior to diagnosis, respectively. This suggests that diagnosis often occurs when there is more multisystem involvement and more engagement with the healthcare system. When analyzed based on specific myopathy-related presentations (e.g., impaired gait or mobility, fatigue, myalgia), 36% of patients with possible PMM (per MD ICD-10 code) had moderate-to-severe presentations, as indicated by inpatient admission or myopathy-related complications (e.g., rhabdomyolysis). Healthcare resource utilization was high, including increased specialist engagement, with the majority of patients seeing a neurologist an average of ~6 times per year. This patient analysis confirms that PMM encompasses a broad spectrum of clinical manifestations that require utilization of extensive healthcare services. Moreover, they underscore the need for more health care provider education about the potential manifestations and multiorgan dysfunctions indicative of PMM, along with information about the types of assessments (e.g., lung function, endurance tests, genetic screening) that can help clinicians make an earlier clinical and genetic confirmatory diagnosis and provide appropriate management.

Abstract #: 2023PA-0000000210

Presenter: Mai Sirimanne

From Clinical Manifestations of Primary Mitochondrial Myopathies (PMM) to Diagnosis: Results from a Patient Journey Analysis Shows Limited Utilization of Genetic Testing

Sirimanne M^1*, Kates J¹, Saikumar S², Warner M³, Shah S², Lovink A², and Yuqing X²

¹Reneo Pharmaceuticals, Inc., Irvine, California, USA; ²Trinity Life Sciences LLC, Waltham, Massachusetts, USA; ³Commercial Rx, Inc., Corona del Mar, California, USA

*msirimanne@reneopharma.com

Primary mitochondrial myopathies (PMM) are a group of disabling and underdiagnosed rare genetic disorders that are usually characterized by musculoskeletal manifestations (e.g., weakness, fatigue, cramping, paralysis, pain) as well as multi-organ dysfunction, including neurologic, cardiac, and endocrinological problems. While the path to PMM diagnosis begins with identifying these manifestations and dysfunctions, their presentations are highly heterogenous, which can make diagnosis very difficult and delay a confirmatory diagnosis based on genetic testing. To better understand the path to diagnosis, analyses were conducted using Komodo closed-claims data for US patients who had a claim between 2016-2021. Due to the absence of a PMM-specific ICD-10 diagnosis code, a stepwise approach was needed to identify patients with suspected PMM. This entailed using ICD-10 diagnosis codes that were specific to a mitochondrial disorder (MD), including chronic progressive external ophthalmoplegia, Kearns-Sayre Syndrome, and Leigh Syndrome. The analysis sample was limited to patients who had ⩾1 myopathy claim, which represented >70% of patients in the initial sample. Patients who had secondary MD ICD-10 code were excluded. The patients included for analysis (N=3.7K) engaged with multiple clinicians, including primary care providers (~78%) who often serve as gatekeepers to patient care, along with a broad range of specialists (neurologists ~50%, cardiologists ~50%, gastroenterologists ~34%, ophthalmologists ~33%). The pathway to diagnosis included a marked increase in physician visits, with 44% of patients visiting 3+ specialists in the 12 months prior to diagnosis as compared to 28% in the 24-36 months prior to diagnosis. While engagement with multiple specialties was high, the involvement of geneticists pre- and post-diagnosis was remarkably low (~10% and 14%, respectively), as was the use of confirmatory genetic testing (~12%). These results highlight a significant gap in the diagnostic journey, since low genetic testing—whether due to limited awareness, interest, or access—creates a suboptimal and burdensome diagnostic approach for patients with PMM. With the recent technical advancements in gene sequencing and more investigational agents for PMM in clinical trials, the low utilization of genetic testing in this real-world cohort highlights the need for expanded education and improved patient access to enable a confirmatory diagnosis of PMM.

Abstract #: 2023PA-0000000211

Presenter: Chad Glasser

Results from a Phase 2a study evaluating zagociguat, a CNS-penetrant sGC stimulator, in adults with Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like Episodes (MELAS)

Glasser C^1*, Karaa A², Falk MJ^3,4, Goldstein A^3,4, Hirano M⁵, Wilson P¹, Chickering J¹, Nguyen S³, King J¹, Savard M¹, Winrow C¹, Kinon B¹

¹Cyclerion Therapeutics, Cambridge, MA, USA; ²Department of Genetics, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; ³Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA; ⁴Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; ⁵Department of Neurology, Columbia University Irving Medical Center, New York, NY, USA

*cglasser@cyclerion.com

Introduction: Zagociguat, previously known as IW-6463 and CY6463, is an orally administered central nervous system (CNS)-penetrant positive allosteric modulator of soluble guanylate cyclase (sGC), a signaling enzyme that catalyzes the formation of cyclic guanosine 3’,5’-monophosphate (cGMP) from guanosine triphosphate (GTP) in response to nitric oxide (NO) binding. Intracellular cGMP regulates vascular tone, regional blood flow, and inflammation, and has been implicated in neuronal survival and cognitive function. Additionally, the NO signaling pathway is critical for mitochondrial function and biogenesis, and in some mitochondrial diseases, including MELAS, NO pathway dysregulation contributes to impaired cerebral blood flow (CBF), oxidative stress, inflammation, and metabolic crises.

Methods: The effects of 29 days of once-daily zagociguat (15mg) were evaluated in a Phase 2a open-label study in adults with MELAS (NCT04475549). Key eligibility criteria included: clinical features of MELAS, prior genetic confirmation of the syndrome consistent with a known disease-causing mutation, plasma lactate ⩾1 mmol/L, and stable chronic medications. Safety, the primary objective, was assessed via adverse event (AE) reporting. Key exploratory endpoints included: changes in plasma biomarkers of mitochondrial dysfunction, endothelial function, and inflammation; zagociguat concentrations in plasma and CSF; and changes in neuroimaging measures of cerebral blood flow and fMRI-BOLD signal.

Results: Eight participants across a range of disease severities were enrolled; 6 of 8 were taking oral NO precursors, arginine or citrulline, daily. Zagociguat was well-tolerated; no serious AEs or treatment discontinuations due to AEs were reported. Zagociguat concentrations in the CSF and plasma were consistent with findings in healthy subjects. Reductions in lactate were observed in 6 of 8 participants and ranged from 7% to 46%. Reductions in GDF-15 were observed in 4 of 8 participants with greatest reductions (up to 39%) in participants with higher baseline concentrations. Changes in these biomarkers of mitochondrial dysfunction were strongly correlated with each other and also correlated with zagociguat plasma concentrations at the end of treatment. Increases in cerebral blood flow were observed in 5 of 8 participants and ranged from 19% to 60%. Increased occipital response to a visual stimulus, enhanced resting state functional connectivity, and impacts across a panel of inflammatory and endothelial biomarkers (many linked to the pathophysiology of MELAS), were also observed.

Conclusion: Zagociguat was well-tolerated and had impacts on multiple disease-relevant objective biomarkers in participants with MELAS on standard-of-care treatments. Additional studies are warranted to further evaluate zagociguat as a potential treatment for patients with MELAS and other mitochondrial diseases.

Abstract #: 2023PA-0000000213

Presenter: Gerardo Piroli

Characterization of a Novel UQCRC1 Variant in a Patient with Progressive Weakness, Pain and Sleep Issues Reveal a Functional Mitochondrial Defect

Piroli GG^1*, Holloway L², Linebaugh E², Skinner C², Skinner S², Frizzell N¹, Steet R²

¹Pharmacology, Physiology & Neuroscience, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Columbia SC 29209; ²Greenwood Genetic Center, Greenwood, SC 29646

*gpiroli@uscmed.sc.edu (*Corresponding author’s email)

UQCRC1 encodes for a subunit of the mitochondrial respiration Complex III. Mutations in UQCRC1 have been suggested to cause autosomal dominant Parkinson’s disease with polyneuropathy. Here we report a patient (P1) and his father (P2) who bear a heterozygous variant of uncertain significance in UQCRC1 (c.302C>T; p.Thr101Ile). This variant has been previously reported in the gnomAD database; however, the frequency data provided is insufficient to deem this a benign alteration. The patient (male in his mid 30s) has progressive weakness, pain, and sleep issues as primary concerns. The father does not exhibit these same symptoms but reports an inability to gain muscle mass despite regular exercise and physical labor. Lymphoblast lines were generated from the patient and father and used to functionally address the significance of the missense variant. Western blot analysis did not reveal defects in the steady-state abundance of the UQCRC1 protein in the patient or father when compared to healthy, age-matched controls (C1, C2), nor was any significant reduction in representative proteins of the different mitochondrial complexes observed. Further functional studies investigating effects on mitochondrial respiration demonstrated decreases in the basal oxygen consumption rate (OCR) in P1 and P2 vs. their respective controls, with the lowest OCR levels observed in P1. Pronounced decreases in maximal uncoupled respiration and spare respiratory capacity were also detected in P1 and P2 vs. healthy controls. The total rate of ATP production as well as the rate of ATP derived from glycolysis and oxidative phosphorylation decreased in P1 and P2 vs. their respective controls. In addition, ATP production was lower in P1 vs. P2, with P1/P2 ratios of 0.48 for total ATP, 0.57 for ATP from glycolysis, and 0.28 for ATP from oxidative phosphorylation. Citrate synthase activity, used to normalize mitochondrial content, showed no differences among the groups. We also challenged lymphoblasts with lipopolysaccharide (LPS, 100 ng/ml for 18 h) and measured the extracellular acidification rate (ECAR) following the sequential addition of glucose, oligomycin and 2-deoxyglucose. The healthy control lymphoblasts responded to the LPS challenge as expected with increased ECAR corresponding to glycolysis, glycolytic capacity and glycolytic reserve. In contrast, the lymphoblasts from P1 and P2 showed limited response to the LPS challenge. The data confirm functional mitochondrial respiratory deficits in the UQCRC1 lymphoblasts, with pronounced reductions in OXPHOS capacity, despite an increased contribution of glycolysis to total ATP levels. The limited ability to increase glycolytic capacity in response to a stressor such as LPS indicates impaired metabolic flexibility. Overall, the metabolic poise of the UQCRC1 mutant lymphoblasts differed with P1 showing more pronounced deficits, potentially contributing to the variable clinical presentations of the patient and father.

Abstract #: 2023PA-0000000214

Presenter: Bibekananda Kar

Enhanced Mitochondrial Base Editing System for Near Complete Mitochondrial Genome Editing in Human Primary Cells Suitable for Disease Modeling and for Potential Therapeutic Applications

Kar B¹*, Thulung LR¹, Castillo SR^{1, 2}, Sabharwal A¹, Zhu M³, Tong Y³, Gong S³, Clark KJ¹, Ekker SC¹

¹Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA; ²Mayo Clinic Graduate School of Biomedical Sciences, Virology and Gene Therapy Track, Mayo Clinic, Rochester, MN 55905, USA; ³Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53706 USA

*kar.bibeknanda@mayo.edu., ekker.stephen@mayo.edu

Mitochondria, as semi-autonomous organelles with unique genetics, present novel experimental and clinical challenges. For example, the consequences of altered mitochondrial physiology due to point mutations can be evident in a tissue-specific manner or as a multi-systemic presentation, often with diverse or highly specific clinical outcomes. Considerable recent work has established new mitochondrial genome editing tools; however, achieving sufficient gene editing in primary cells for effective disease modeling or therapeutic applications remains a challenge, as a threshold percentage of variant mtDNA must be surpassed for altered function to reveal subsequent disease phenotypes. Our group recently established the FUSXTBE mitochondrial base editing system, which initially focused on TC-to-TT variations in zebrafish in vivo and human cells in vitro. Our animal model work resulted in near-complete (>75%) mtDNA editing, setting the precedent for achieving a similar goal in human primary cells. We report here an improved and universal FUSXTBE system with enhanced and multiple C-to-T and A-to-G base editing functionality in human primary cells. Single dose delivery of these editors demonstrated near-complete gene editing events in human primary cells without any selection. By using this new platform, we were able to establish different mitochondrial disease models with phenotypic outcomes. In addition, we have adopted virus-free delivery of these enhanced mitochondrial base editors for future in vivo studies. These improvements enable more advanced preclinical research and could serve as the foundation for a potential new therapeutic platform.

Abstract #: 2023PA-0000000215

Presenter: Ankit Sabharwal

Fishing for Cures: Zebrafish as a Pioneering In Vivo Model to Help Solve Mitochondrial Medicine Odysseys

Sabharwal A¹, Savage K.S¹, Kar B¹, Thulung L.R¹, Daby C.L¹, Clark K.J¹, Ekker S.C.^1*

¹Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA

*Corresponding author: Ekker S.C. - ekker.stephen@mayo.edu

Mitochondria play an important but still largely mysterious role in human physiology, as demonstrated by the enormous biological variation and diverse disorders in patients with mitochondrial disease. These include neurologic, cardiac, endocrine, kidney, visual, hearing, blood, and skeletal muscle systems. Understanding how mitochondria function in normal biology and how human mitochondrial DNA variations contribute to health and disease has been hampered by a few effective approaches to manipulate mtDNA and a lack of existing animal models. The establishment of CRISPR-free, TALE-derived base editors demonstrated that targeted C to T (or G to A) and later A to G (or T to C) mtDNA sequence changes were possible in human cells and in initial mouse model work. To enhance the accessibility of this new editing tool and to enable rapid testing in new in vivo models, we developed the next-generation FusX TALE Base Editor (FusXTBE) system incorporating cytosine and adenosine base editors. FusX is a one-step TALE assembly system that has been used to make an array of functional TALENs that work from flies to fish to human cells. The ability to make animal models of specific sequence variants is a function of both sequence conservation as well as the potential gene editing capabilities of the molecular toolbox. The mitochondrial genome is highly conserved in all vertebrates, for example, the zebrafish mitochondrial genome is nearly identical in size (16kb+) and encodes the same complement of genes that are organized in the same order as the human mitochondrial genome. To explore the use of TALE mitochondrial base editing technology to model mutations, we first targeted one of the mt-tRNA genes, the human ortholog of which has been implicated in the progression of various mitochondrial disorders such as MELAS, renal dysfunction, and other clinical indications. Here in this study, zebrafish embryos were used as a pioneering in vivo test system of mtDNA-encoded mitochondrial dysfunction. Injected animals with FusXTBE harbor edits in mtDNA loci with over 90% editing efficiency in F0 animals as a novel example of majority mtDNA heteroplasmy induction in an in vivo system. We have extended this work to explore detailed germline transmission analyses with near-complete mutant mtDNA heteroplasmy in F1 animals (over 80%). Zebrafish mtDNA mutants not only show impaired mitochondrial bioenergetics but have an altered transcriptomic signature pertaining to respiration and cholesterol synthesis pathways. Interestingly, zebrafish mutants display differential segregation of pathogenic edits with distinct transcriptomic signatures. We have also prioritized pathogenic mtDNA loci for conservation at the amino acid (protein-coding) or nucleotide (for tRNA) level to enable to model/correct pathogenic edits in the mitochondrial genome. We have established the first zebrafish model of MELAS, and we are making a series of additional validated zebrafish lines harboring designer mtDNA variants suitable for hypothesis testing as well as discovery science. The FUSXTBE molecular toolbox will also be optimized for utility in helping generate other animal models from work by mitochondrial scientists in the field. Together, these gene editors and in vivo avatars will enable new approaches for diagnoses and therapies for these terrible diseases.

Abstract #: 2023PA-0000000216

Presenter: Lana Sheta

The phenotypic severity of overlapping mitochondrial deletions is positively associated with heteroplasmy level in two individuals

Sheta LM^1*

¹Variantyx, Inc, USA

*lana.sheta@variantyx.com

Mitochondrial disorders are a heterogeneous group of diseases that can present with a wide range of clinical manifestations, including hearing impairment, vision loss, muscle weakness, and neurological deficits. Large-scale mitochondrial deletion syndromes generally comprise three overlapping phenotypes including Kearns-Sayre syndrome, Pearson syndrome, and progressive external ophthalmoplegia (PEO), where heteroplasmy level generally correlates with phenotypic severity. Here, we examine two individuals with overlapping large-scale mitochondrial deletions, detected by whole genome sequencing (WGS) in diagnostic testing, and explore the relationship between their clinical presentation and heteroplasmy level.

Case 1 was a 19-year-old individual with a 2.30 kb mitochondrial deletion (m.12112_14416del) that included the MT-ND4, MT-ND5, MT-ND6, MT-TH, MT-TL2, and MT-TS2 genes. This deletion was detected at 20% heteroplasmy in the proband's saliva. This deletion was not detected in the mother’s saliva sample, suggesting that it arose de novo in the proband. The patient presented for diagnostic genetic testing with a history of multisystem involvement, including hearing impairment, rod-cone dystrophy, tubulointerstitial nephritis, stage 5 chronic kidney disease necessitation renal transplantation at age 11, type I diabetes mellitus, and hypertrichosis. The patient had an unremarkable birth and early childhood history, and met developmental milestones on time. Family history was negative for hearing loss and vision impairment, and consanguinity was denied. Prior testing included a negative mitochondrial genome deletion and duplication analysis; normal urine organic acid analysis; and abnormal mitochondrial electron transport chain testing on muscle biopsy, which demonstrated abnormally low activity in the rotenone sensitive complex I.

Case 2 was a 44-year-old female with a 1% heteroplasmy mitochondrial deletion (m.8482_ 13446del) detected in saliva. It spanned 4.96 kb and included the MT-ATP6, MT-CO3, MT-ND3, MT-ND4, MT-ND4L, MT-TG, MT-TH, MT-TL2, MT-TR, and MT-TS2 genes. This alteration is known as the common deletion, and it was first described in an individual with PEO. Maternal family members were unavailable for testing. She presented with progressive external ophthalmoplegia, unilateral ptosis, diplopia, migraine, palpitations, and attention deficit hyperactivity disorder with onset in early adulthood. Family history was negative for PEO. Previous testing of the nuclear genome was non-diagnostic.

The clinical presentation of Case 1 is atypical for mitochondrial deletion syndromes, as ophthalmoplegia, cardiac conduction abnormalities, hematological phenotypes, significant muscle weakness, and ragged red fibers were not identified. Instead, this individual had rod-cone dystrophy, severe renal failure, and notable endocrine dysfunction. Conversely, Case 2 had a classic PEO presentation. While the deletion identified in Case 2 was larger, its lower level of heteroplasmy (1%) correlated with the comparatively milder phenotype compared to Case 1 (20% heteroplasmy).

In conclusion, these cases further support the understanding that the phenotypic spectrum associated with mitochondrial deletions is wide, and severity correlates with degree of heteroplasmy. Additionally, these findings demonstrate the importance of highly sensitive WGS in the detection of heteroplasmic mitochondrial deletions, particularly in cases where previous genetic testing has been non-diagnostic, and highlight the need for further study to better understand the complex relationship between genotype and phenotype in mitochondrial disease.

Abstract #: 2023PA-0000000218

Presenter: Norma Frizzell

Linking Altered Microglial Metabolism To An Impaired Inflammatory Response In Leigh Syndrome

McCain RS, Smith HH, Swaminathan S, Piroli GG, and Frizzell N

Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia SC 29209

norma.frizzell@uscmed.sc.edu

The genetic mitochondrial disease Leigh syndrome is a progressive encephalopathy associated with bilateral necrotizing lesions of the brainstem and basal ganglia. Clinically the patients exhibit periodic lactic acidosis, a failure to thrive, respiratory failure, and are at risk of early death. Genetic defects in components of the electron transport chain, including Complex I, are responsible for the bioenergetic defect associated with the disease. The NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4, a Complex I assembly factor) knockout (KO) mouse is an established mouse model of Leigh Syndrome. The NDUFS4 KO mouse manifests many of the biochemical and clinical aspects of Leigh syndrome, including microglial accumulation in lesioned brain regions.

Following recent reports of a hyperinflammatory state in models of mitochondrial disease, we examined the immunometabolism and inflammatory profile of peritoneal macrophages isolated from the NDUFS4 KO mouse. Lipopolysaccharide (LPS) challenge enhanced glycolytic metabolism in both wild type (WT) and KO mice, but NDUFS4 KO mice had a lower response and limited glycolytic reserve, indicating impaired metabolic flexibility in response to a challenge. Itaconate, derived from the TCA metabolite cis-aconitate, is produced in response to LPS challenge and acts to suppress pro-inflammatory activity. WT macrophages showed pronounced increases in itaconate production (>40-fold) in response to LPS. In contrast, the NDUFS4 KO mice exhibited an ~10-fold increase in itaconate, indicating that Complex I deficiency impaired production of this immunomodulatory metabolite in peritoneal macrophages. LPS challenge also resulted in an impaired induction of several pro- and anti-inflammatory cytokines and reduced phagocytic uptake in NDUFS4 KO vs. WT macrophages. Overall, these studies demonstrate a blunted immunometabolic and inflammatory response to LPS in the isolated NDUFS4 KO macrophages, which may also reflect impaired immunomodulatory responses in the microglia within brain lesions.

Abstract #: 2023PA-0000000219

Presenter: Suzanne Scheller

Using Creighton Model FertilityCare™System Biomarkers to Evaluate and Treat Hormone Dysfunction in Inflammatory Disease, Endometriosis, with Case Study

Scheller, SB*

Ethics & Biotechnology, Graduate Student, John Paul II Institute, United States

*schellers@cua.edu

Hormone fluctuations are known to contribute to the severity of symptoms in patients with mitochondrial and inflammatory disease, including endometriosis. However, a precise picture of the mechanisms underlying volatile processes involved can be elusive. For example, although endometrial tissue should form solely inside the uterus, in patients with endometriosis, it grows in other places. In such cases, the programmed cell death process is disrupted, indicating that endometriosis is in part a mitochondrial problem. Hormones also play a significant role because they drive the thickening and bleeding of endometrial tissues. Yet, even though this condition affects one in ten women, in the U.S., it can remain undiagnosed for up to 6 years from the symptom onset. These affected women could thus greatly benefit from the Creighton Model Fertility Care System^TM (CrMS) and the woman-specific feedback it provides. The standardized CrMS notations allow the female cycle to be charted and interpreted as a vital sign. Cycle comparisons with normal patterns and values yield meaningful information about hormone health or dysfunction. Although invisible to any form of radiography, with CrMS biomarkers, endometriosis becomes suspect in only three cycles, thus shortening the time to diagnosis to under a year. This review focuses on the CrMS biomarkers developed at St. Paul VI Institute for the Study of Human Reproduction to explore the interplay between hormonal and mitochondrial dysfunction in a patient with multiple system involvement, including diagnoses of bipolar 1, miscarriage, mitochondrial dysfunction and endometriosis. The process is explained by examining two biomarkers—unusual bleeding and the post-peak phase length with blood and genetic test results for hormonal and mitochondrial aberrations. In this patient, progesterone deficiencies were first identified in the fourth week of her third pregnancy after a prior miscarriage and were also present later during perimenopause. Targeted progesterone therapy protocols, unique to the medical science associated with CrMS called NaProTECHNOLOGY^® (NPT), were applied and led to the prevention of a miscarriage and alleviation of premenstrual dysphoric disorder symptoms, as well as remission of endometriosis and bipolar symptoms. These exceptionally positive outcomes suggest the presence of connections between hormonal and mitochondrial dysfunction. Mitochondria produce the progesterone that supports the luteal phase of the cycle, influences the health of the subsequent cycle, as well as sustains pregnancy. Therefore, this case study stimulates discussion about the feedback loops in female patients, involving stressors, hormone levels, and mitochondrial roles in hormone activity. A collaboration between members of the NPT and mitochondrial medicine communities would help elucidate biofeedback driving the lack of apoptosis in endometriosis as well as the relationships between hormone and mitochondrial dysfunction. Such collaboration would also lead to the improvement of women’s health prior to conception, thus having a positive influence on the follicle and egg selection and subsequently on conception and implantation processes. These interventions could 1) favor positive epigenetic outcomes for the offspring, including potentially lower detrimental heteroplasmy rates that affect the mitochondrial health of the offspring and 2) interrupt the disease processes and bring potentially timely healing to both mother and child.

Abstract #: 2023PA-0000000221

Presenter: Kaylee Steiner

Mitochondrial Trans-2-Enoyl Coenzyme A Reductase (Mecr) Regulates CD4⁺ T Cell Function

*Steiner KK^1,2, Young AC², Patterson AR², Chi C ², Sugiura A², Rathmell JC²

¹Cancer Biology Graduate Program, Vanderbilt University, USA; ²Pathology, Microbiology, and Immunology Department Vanderbilt University Medical Center, USA;

*kaykee.k.steiner@vanderbilt.edu

Many inflammatory diseases and cancer can be driven by dysregulation of T helper cells (Th cells). We have previously shown that manipulation of metabolic pathways can affect Th cell differentiation and disease development. We developed a CRISPR library to investigate the role of lipid metabolism in survival and proliferation of Th cells. In pooled genetic screens using in vivo models of allergic airway disease and inflammatory bowel disease, CD4⁺ T cells were transduced with a lipid metabolism library and adoptively transferred into Rag1 ^-/- mice. After disease development, the relative abundance of each guide RNA was determined in lung-infiltrating CD4⁺ T cells to identify immunometabolic regulators of CD4⁺ T cell recruitment and persistence in inflammation. Mitochondrial trans-2-enoyl-coenzyme A reductase (Mecr) was a significantly depleted gene, demonstrating a potential role in T cell-mediated lung inflammation. Interestingly, the function of Mecr in immune cells is unknown and recessive mutations in humans cause an inborn error of metabolic disorder. Therefore, further studies used CRISPR/Cas9 to test the role of Mecr in T cell function. When tested in a side-by-side Th17-transfer inflammatory bowel disease model, the proportion of Mecr-knockout cells was decreased compared to a non-targeting control in the spleens, mesenteric lymph nodes, lamina propria, and intra-epithelial lymphocytes in addition to lower IFNg and Tbet expression. Data also show that Mecr-knockout in CD4⁺ T cells in vivo have increased cell death by apoptosis. Together, these results show that Mecr plays an important role in T cell immunometabolism. Further studies are focused on elucidating its role in inflammatory diseases in different T cell subsets.

Abstract #: 2023PA-0000000224

Presenter: Dmitry Temiakov

Molecular Basis for Maternal Inheritance of Human Mitochondrial DNA

Lee W.¹†, Zamudio-Ochoa A.,¹†, Buchel G.¹†, Podlesniy P.², Marti Gutierrez N.³, Puigros M.², Calderon A.², Tang H.Y.⁴, .Li L.⁵, Mikhalchenko A.³, Koski A.³, Trullas R.², Mitalipov S.³, and Temiakov D.¹*

¹Department of Biochemistry and Molecular Biology, Thomas Jefferson University; 1020 Locust St, Philadelphia 19107, USA; ²Neurobiology Unit, Institut d’Investigacions Biomèdiques de Barcelona (IIBB-CSIC-IDIBAPS) and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED); Barcelona, 08036, Spain, ³Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University; 3303 W Bond Avenue, Portland, Oregon 97239, USA; ⁴The Wistar Institute, Philadelphia, 3601 Spruce Street, Philadelphia, PA 19104, USA, ⁵Department of Pathology, Thomas Jefferson University; 1020 Locust St, Philadelphia 19107, USA

*Corresponding author. Email:dmitry.temiakov@jefferson.edu; †These authors contributed equally to this work

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy. However, the causative mechanisms of paternal mtDNA elimination have not been defined. We found that mitochondria in human spermatozoa are devoid of mtDNA and lack mitochondrial transcription factor A (TFAM), the major nucleoid protein required to protect, maintain, and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial pre-sequence, ordinarily removed upon mitochondrial import. Phosphorylation of this pre-sequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM re-localization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mitochondrial DNA, thereby explaining maternal inheritance in this species.

Abstract #: 2023PA-0000000226

Presenter: Cameron Menezes

Determining the In Vivo Impact of Mitochondrial Energy Loss in the Liver

Cameron J Menezes¹ Xun Wang, PhD², Prashant Mishra, PhD³

^1,2UT Southwestern Medical Center, Children's Medical Center Research Institute ³UT Southwestern Medical Center, Children's Medical Center Research Institute, Department of Pediatrics

Mitochondrial energetic dysfunction is implicated in many common human diseases, including conditions impacting the liver such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and liver failure. In cultured cells, loss of mitochondrial ATP production is well-tolerated via metabolic compensation from glycolysis-derived ATP. In contrast, the precise consequences of loss of mitochondrial energetics in the mammalian liver are largely unknown. To date our understanding of the roles of mitochondrial complex V (mCV), or ATP synthase, has arisen primarily from in vitro cultured cells or yeast studies. These seminal results yielded structural and functional insights into mCV’s role as the canonical “powerhouse of the cell”, but are unable to delineate the fundamental dependence of various cellular processes on mitochondrial ATP production. To address this, we have developed and validated the first engineered mouse model which conditionally targets mCV, by inserting loxP sites surrounding exon 2 of the α subunit (ATP5F1A). Biochemical analysis revealed a complete loss of ATP synthesis activity in mutant tissue. Administration of a liver specific AAV8 virus encoding Cre recombinase (or GFP) at 8 weeks of age resulted in impaired survival, precipitous weight loss, and inhibition of gluconeogenesis activity. Terminal tissue and blood collection demonstrated increases in markers of liver damage (ALT/AST), decreases in hepatic TCA cycle acyl-CoA species and adenosine nucleotides, and decreases in circulating albumin. Ultrastructural analysis reveals significant deficits in cristae formation in ATP5FA1 deficient cells. Taken together, these results demonstrate that mitochondrial energy production is required for homeostatic function of the murine liver, including physiologic maintenance of circulating glucose and protein levels and organelle structure, and implicates mitochondrial dysfunction as a contributing factor to liver failure.

Abstract #: 2023PA-0000000230

Presenter: Ernst-Bernhard Kayser

Temporary Treatment of Leigh Syndrome Reveals Clues to its Pathogenesis

Ernst-Bernhard Kayser, Yihan Chen, Kyung Yeon Park, Michael Mulholland, John Snell, Kiheon Suh, Simon Johnson

Seattle Children’s Research Institute, Seattle WA, Center for Integrative Brain Research

The Ndufs4KO mouse is an established model for Leigh Syndrome. The untreated KO mouse shows normal behavior and gains weight until age P38 (postnatal day 38). At this developmental stage cachexia sets in followed by signs of brain damage (e.g. clasping behavior), and ultimately death at P65 (median age). Several treatments are known to improve health and lifespan when administered continuously.

We investigated the possibility that temporary treatment, given only during developmental time windows covering the critical onset age of P38, may convey lasting benefits. Here, we report results for 3 types of interventions: 1. Daily IP injection of an albumin adsorbed formulation of the mTOR inhibitor rapamycin (ABI-009, 9mg/kg/d) from P30-P40 or P26-P40. 2. Hypoxia (air with 11% O2) from P26-40 or P21-P60. 3. Feeding of the CSF1R inhibitor pexidartinib from P21-P40. Disease progression was monitored by assessing weight, clasping behavior and survival.

Only rapamycin conveyed moderate sustained benefits: Suppression of cachexia, delay of clasping and median survival. E.g. treatment during P26-P40 increased median survival to P97. While in hypoxia mice remained very healthy but disease set in and progressed extremely rapid after shift to normal air leading to accelerated death: P54 and P65 for treatment windows P26-P40 and P21-P60 respectively. Pexidartinib lead to a 2 week increase of healthspan (delay of cachexia after treatment stop). Afterwards disease onset was sudden, quickly followed by death leaving no survival benefit over no treatment at all.

It was remarkable that response to rapamycin treatment is very variable between individual KO mice while hypoxia and pexidartinib withdrawal has a synchronizing effect on disease progression. Other studies in our lab have identified a macrophage mediated autoimmune response as a major driver of disease progression. We therefore postulate that temporary rapamycin treatment may permanently modify the immune system while hypoxia and pexidartinib can only suppress it acutely. The delayed disease onset after pexidartinib probably reflects the time necessary for the depleted macrophage population to recover.

Abstract #: 2023PA-0000000236

Presenter: Jonathan Dietz, PhD

The Role of C. elegans Metaxins in Mitochondrial Homeostasis

Jonathan V. Dietz, Eunchan Park, Nathaly Salazar-Vasquez, Nanci Kane, Carol Nowlen, and Christopher Rongo

Waksman Institute, Rutgers University and Department of Genetics, Rutgers University

Mitochondria are critical for neuronal function and health, as they are the primary supplier of energy and calcium storage for neurons. Mitochondrial dynamics – fusion, fission, and motility – facilitate energy production and calcium buffering by mitochondria at specific subcellular sites within neurons. Disturbances in mitochondrial function or dynamics contribute to various neurodegenerative disorders. Using a forward genetic screen in C. elegans searching for novel mutants defective in neuronal mitochondrial dynamics, we found that mutations in metaxin 1 (MTX-1), metaxin 2 (MTX-2), and VDAC-1 resulted in fewer mitochondria in C. elegans interneuron dendrites. Mammalian metaxin homologs interact with SAM50 to form the sorting and assembly machinery (SAM) complex, which mediates β-barrel protein assembly in the mitochondrial outer membrane (MOM). VDAC-1 is a highly conserved SAM complex substrate that acts as a channel for metabolites across the MOM. We hypothesize that the metaxins promote mitochondrial motility along C. elegans interneuron dendrites by mediating assembly of VDAC-1 in the MOM. Mutants for mtx-1, mtx-2, and vdac-1 are viable but have reduced lifespans. We found that the mitochondrial unfolded protein response (UPRmt) was activated in mtx-2 and vdac-1 mutants, resulting in heat stress resistance and mitohormesis. We are currently investigating the role of C. elegans MTX-1 and MTX-2 in MOM β-barrel protein (VDAC-1) assembly and how that impacts neuron integrity.

Abstract #: 2023PA-0000000237

Presenter: Conor Ronayne, PhD

Mitochondrial Ribosome Signaling and Survival Mechanisms in Mitochondrial Disease

Conor T. Ronayne^1,2, Thomas D. Jackson^1,2, Christopher F. Bennett^1,2, Elizabeth A. Perry^1,2, Noa Kantorovic^1,2 and Pere Puigserver^1,2

¹Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115 and ²Department of Cell Biology, Harvard Medical School, Boston, MA 02115

Mitochondrial bioenergetic defects occur in mitochondrial diseases or in conditions associated with mitochondrial dysfunction. These defects cause a series of metabolic and cellular dysregulation including cell death and inflammatory processes resulting in chronic and potentially fatal tissue damage. Among these defects are mitochondrial mutations in genes encoded by the nuclear or mitochondrial genome causing diseases without medical treatments. The mechanistic basis of these pathologies includes imbalance of redox and inflammatory processes that are exacerbated in tissues such as brain or skeletal muscle causing neuropathies or myopathies, respectively. In this regard, our laboratory has developed chemical and genetic screening platforms using mitochondrial disease mutant cells and has identified genes and small molecules that rescue mitochondrial deficiencies. One of the hits of these high throughput screens were a series of antibiotics including tetracyclines that target mitochondrial ribosomes (mitoribosomes) and inhibit mitochondrial protein synthesis. Tetracyclines were validated in mitochondrial complex I (Ndufs4) KO mice (a model of Leigh syndrome mitochondrial disease) indicating that partial inhibition of mitochondrial translation rescues cell survival and inflammation associated with mitochondrial bioenergetic defects. However, the potential signaling mechanisms and importantly their role in cell survival of mitochondrial mutant cells is entirely unknown. We propose in this application a model that mitoribosome stalling induced by tetracyclines initiates a signaling mechanism that causes cell survival or death in the context of mitochondrial bioenergetic defects. The identification of this signaling and the effects of tetracyclines will be investigated for the development of new generation agents for the treatment of mitochondrial diseases.

Abstract #: 2023PA-0000000238

Implementing a Novel No-Cost Genetic Testing Program for Patients with a High Likelihood of Having Primary Mitochondrial Disease

Philip E. Yeske, PhD¹ and Lukas Lange, PhD²

¹United Mitochondrial Disease Foundation, Pittsburgh, PA; ²Probably Genetic, San Francisco, CA, USA

Objective: Facilitate genetic diagnosis of patients suspected of having primary mitochondrial disease using a patient-initiated remote testing program.

Background: Primary mitochondrial disease (PMD) is a heterogeneous collection of rare inherited genetic disorders. As such, a genetic test is the quickest and most accurate way to confirm PMD and can lead to better medical care, participation in clinical trials and the comfort of knowing your condition. Despite this, many PMD patients do not have a genetic diagnosis, with testing often costly and difficult to access. To address this challenging diagnostic journey the United Mitochondrial Disease Foundation partnered with Probably Genetic to launch a novel, no-cost genetic testing program for potential PMD patients.

Methods: Probably Genetic’s proprietary machine learning-driven Symptom Checker technology was first trained on genetically confirmed cases of PMD and then used to identify US-based patients with a high likelihood of mitochondrial disease based on patient self-reported phenotypes. Genetic testing (whole exome sequencing) was then carried out completely remotely via telemedicine, with all test requests and genetic counseling facilitated by an independent physician network.

Results: Within three months, over 1,500 submissions were received. Dozens of different symptoms were reported by the program participants, with fatigue, digestive issues, muscle weakness, exercise intolerance and cognitive issues reported by over 70% of cases. In addition to suspected mitochondrial disease, over 25 unique differential diagnoses were reported by the patients prior to testing. Participants listed over 1,600 treating physicians, with the majority specializing in internal medicine, neuropsychiatry, family practice or pediatric medicine. 361 patients were offered testing, 226 (63%) of which were claimed. 215/226 (96%) of biosamples (buccal swab) were returned to the testing lab, demonstrating the drive of the patients to end their diagnostic journey. 146/215 (67%) had findings included with their genetic test report and 31/146 (37%) had causative variants of PMD reported. Based on causative findings and compared to published clinical cohorts, this program achieved a competitive overall screening rate (31/215, 14% vs. 20-54%).

Conclusions: A pilot program to enable patients to facilitate their own genetic diagnosis of PMD was carried out. Analysis of the data demonstrates significant potential for the approach, despite the use of a limited genetic testing type (whole exome without mtDNA sequencing) and not using any form of clinical selection (e.g., clinical diagnoses, enzyme tests, PMD score thresholds). This novel pilot program demonstrated the power of patient-initiated remote testing programs and will serve as an excellent model for additional diagnostic initiatives within the mitochondrial disease patient community.

Abstract #: 2023PA-0000000239

Philip E. Yeske, PhD, Nicole Wilson

MitoSHARE: a World-wide Patient-populated Registry for Mitochondrial Disease Patients and their Caregivers

United Mitochondrial Disease Foundation, Pittsburgh, USA

Objective: mitoSHARE is a worldwide patient-populated registry initiative stewarded by the United Mitochondrial Disease Foundation with the goal of advancing scientific research using data gathered from patients and families affected by mitochondrial disease. We aim to identify and characterize mitochondrial disease patients, both minors and adults, on a global scale in a robust research database.

Background: Patient registries are a critical component of therapeutic development, enabling the identification and characterization of a disease patient community. Importantly, patient-populated registries also help to capture the “patient voice” and provide a rich source of data complementary to clinician-populated registries. Launched in March 2022, mitoSHARE recruits caregivers and patients with any type of mitochondrial disease and regardless of diagnostic status. The registry principally aims to advance research and facilitate clinical trial recruitment, but also provides a suite of support tools for both patients and caregivers.

Methods: mitoSHARE is run as an IRB-approved research study. At time of account creation all registrants must first complete an e-consent process for the use of their de-identified data in research. Once consent is obtained, registry participants are presented surveys that collect demographic and diagnostic status data. Participants may also the use the registry as a central repository for electronic health records, curation of genetic testing reports and completion of longitudinal health assessment surveys used to build natural history data. Clinicians are also able to join the platform and, with patient approval, view data from registrants for whom they provide care.

Results: In the first year over 1200 patients and caregivers from 29 countries have started the account creation process with more than three fourths providing consent and entering data into mitoSHARE. To-date 83% of registrants are from the United States with most of the participants between 35-74 years of age. 69% of participants identify as patients and 31% as caregivers (~10% as both). Over 60 different types of clinical and genetic diagnoses are represented in the registry, although nearly 25% of patient participants self-report as “suspected mitochondrial disease”, reinforcing the challenging diagnostic journey many members of the mitochondrial disease community endure. 54% of the patients reported that they have had genetic testing done and 86 of those patients had their genetic test curated through mitoSHARE.

Conclusions: In just one-year mitoSHARE has established itself as an effective tool for identifying and characterizing mitochondrial disease patients and caregivers around the world. Future efforts will include continued patient recruitment and engagement, disease specific surveys, publishing additional longitudinal surveys/patient-reported outcomes as well as collaborating with researchers and industry to facilitate research toward the development of treatments and cures for mitochondrial disease.