Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.

Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.

Interim analyses of a first-in-human phase 1/2 mRNA trial for propionic acidaemia.

Propionic acidaemia is a rare disorder caused by defects in the propionyl-coenzyme A carboxylase α or ß (PCCA or PCCB) subunits that leads to an accumulation of toxic metabolites and to recurrent, life-threatening metabolic decompensation events. Here we report interim analyses of a first-in-human, phase 1/2, open-label, dose-optimization study and an extension study evaluating the safety and efficacy of mRNA-3927, a dual mRNA therapy encoding PCCA and PCCB. As of 31 May 2023, 16 participants were enrolled across 5 dose cohorts. Twelve of the 16 participants completed the dose-optimization study and enrolled in the extension study. A total of 346 intravenous doses of mRNA-3927 were administered over a total of 15.69 person-years of treatment. No dose-limiting toxicities occurred. Treatment-emergent adverse events were reported in 15 out of the 16 (93.8%) participants. Preliminary analysis suggests an increase in the exposure to mRNA-3927 with dose escalation, and a 70% reduction in the risk of metabolic decompensation events among 8 participants who reported them in the 12-month pretreatment period.

Establishment of a non-integrated iPSC line (SDQLCHi043-A) from a male infant with propionic acidemia carrying compound heterozygote mutations in PCCB gene.

In this study, peripheral blood mononuclear cells were contributed from a male infant with propionic acidemia (PA) verified by clinical and genetic diagnosis, who inherited compound heterozygous mutations in the propionyl-CoA carboxylase subunit beta (PCCB) gene. Here, this iPS was generated by non-integrated episomal vectors with SOX2, BCL-XL, OCT4, C-MYC and OCT4. Also, this iPSC line exhibited the morphology of pluripotent stem cells, upward mRNA and protein expression of pluripotency markers, conspicuous in vitro differentiation potency and regular karyotype, and carried PCCB gene mutations, which provided an excellent model for the research and drug screening of PA.

Gene therapy for organic acidemias: Lessons learned from methylmalonic and propionic acidemia.

Organic acidemias (OA) are a group of rare autosomal recessive disorders of intermediary metabolism that result in a systemic elevation of organic acid. Despite optimal dietary and cofactor therapy, OA patients still suffer from potentially lethal metabolic instability and experience long-term multisystemic complications. Severely affected patients can benefit from elective liver transplantation, which restores hepatic enzymatic activity, improves metabolic stability, and provides the theoretical basis for the pursuit of gene therapy as a new treatment for patients. Because of the poor outcomes reported in those with OA, especially methylmalonic and propionic acidemia, multiple gene therapy approaches have been explored in relevant animal models. Here, we review the results of gene therapy experiments performed using MMA and PA mouse models to illustrate experimental paradigms that could be applicable for all forms of OA.

Functional analysis of novel variants identified in cis in the PCCB gene in a patient with propionic acidemia.

Next-generation sequencing has improved the diagnosis of inborn errors of metabolism, allowing rapid confirmation of cases detected by clinical/biochemical studies or newborn screening. The challenge, however, remains for establishing the pathogenicity of the identified variants, especially for novel missense changes or small in-frame deletions. In this work we report a propionic acidemia patient exhibiting a severe neonatal form with coma and hyperammonaemia. Genetic analysis identified the previously described pathogenic PCCB variant p.R512C in the maternal allele and two novel PCCB variants in cis in the paternal allele, p.G246del and p.S322F. Expression analysis in a eukaryotic system confirmed the deleterious effect of the novel missense variant and of the one amino acid deletion, as they both exhibited reduced protein levels and reduced or null PCC activity compared to the wild-type construct. Accordingly, the double mutant resulted in no residual activity. This study increases the knowledge of the genotype-phenotype correlations in the rare disease propionic acidemia and highlights the necessity of functional analysis of novel variants to understand their contribution to disease severity and to accurately classify their pathogenic status. In conclusion, two novel PCCB pathogenic variants have been identified, expanding the current mutational spectrum of propionic acidemia.

A common benign intronic deletion masking a pathogenic deep intronic PCCB variant - genome sequencing and RNA studies to the rescue.

Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by variants in PCCA or PCCB, both sub-units of the propionyl-CoA carboxylase (PCC) enzyme. PCC is required for the catabolism of certain amino acids and odd-chain fatty acids. In its absence, the accumulated toxic metabolites cause metabolic acidosis, neurologic symptoms, multi-organ dysfunction and possible death. The clinical presentation of PA is highly variable, with typical onset in the neonatal or early infantile period. We encountered two families, whose children were diagnosed with PA. Exome sequencing (ES) failed to identify a pathogenic variant, and we proceeded with genome sequencing (GS), demonstrating homozygosity to a deep intronic PCCB variant. RNA analysis established that this variant creates a pseudoexon with a premature stop codon. The parents are variant carriers, though three of them display pseudo-homozygosity due to a common large benign intronic deletion on the second allele. The parental presumed homozygosity merits special attention, as it masked the causative variant at first, which was resolved only by RNA studies. Arriving at a rapid diagnosis, whether biochemical or genetic, can be crucial in directing lifesaving care, concluding the diagnostic odyssey, and allowing the family prenatal testing in subsequent pregnancies. This study demonstrates the power of integrative genetic studies in reaching a diagnosis, utilizing GS and RNA analysis to overcome ES limitations and define pathogenicity. Importantly, it highlights that intronic deletions should be taken into consideration when analyzing genomic data, so that pseudo-homozygosity would not be misinterpreted as true homozygosity, and pathogenic variants will not be mislabeled as benign.

Prevalence of propionic acidemia in China.

Propionic acidemia (PA) is a rare autosomal recessive congenital disease caused by mutations in the PCCA or PCCB genes. Elevated propionylcarnitine, 2-methylcitric acid (2MCA), propionylglycine, glycine and 3-hydroxypropionate can be used to diagnose PA. Early-onset PA can lead to acute deterioration, metabolic acidosis, and hyperammonemia shortly after birth, which can result in high mortality and disability. Late-onset cases of PA have a more heterogeneous clinical spectra, including growth retardation, intellectual disability, seizures, basal ganglia lesions, pancreatitis, cardiomyopathy, arrhythmias, adaptive immune defects, rhabdomyolysis, optic atrophy, hearing loss, premature ovarian failure, and chronic kidney disease. Timely and accurate diagnosis and appropriate treatment are crucial to saving patients' lives and improving their prognosis. Recently, the number of reported PA cases in China has increased due to advanced diagnostic techniques and increased research attention. However, an overview of PA prevalence in China is lacking. Therefore, this review provides an overview of recent advances in the pathogenesis, diagnostic strategies, and treatment of PA, including epidemiological data on PA in China. The most frequent variants among Chinese PA patients are c.2002G > A in PCCA and c.1301C > T in PCCB, which are often associated with severe clinical symptoms. At present, liver transplantation from a living (heterozygous parental) donor is a better option for treating PA in China, especially for those exhibiting a severe metabolic phenotype and/or end-organ dysfunction. However, a comprehensive risk-benefit analysis should be conducted as an integral part of the decision-making process. This review will provide valuable information for the medical care of Chinese patients with PA.

Pathophysiological mechanisms of complications associated with propionic acidemia.

Propionic acidemia (PA) is a genetic metabolic disorder caused by mutations in the mitochondrial enzyme, propionyl-CoA carboxylase (PCC), which is responsible for converting propionyl-CoA to methylmalonyl-CoA for further metabolism in the tricarboxylic acid cycle. When this process is disrupted, propionyl-CoA and its metabolites accumulate, leading to a variety of complications including life-threatening cardiac diseases and other metabolic strokes. While the clinical symptoms and diagnosis of PA are well established, the underlying pathophysiological mechanisms of PA-induced diseases are not fully understood. As a result, there are currently few effective therapies for PA beyond dietary restriction. This review focuses on the pathophysiological mechanisms of the various complications associated with PA, drawing on extensive research and clinical reports. Most research suggests that propionyl-CoA and its metabolites can impair mitochondrial energy metabolism and cause cellular damage by inducing oxidative stress. However, direct evidence from in vivo studies is still lacking. Additionally, elevated levels of ammonia can be toxic, although not all PA patients develop hyperammonemia. The discovery of pathophysiological mechanisms underlying various complications associated with PA can aid in the development of more effective therapeutic treatments. The consequences of elevated odd-chain fatty acids in lipid metabolism and potential gene expression changes mediated by histone propionylation also warrant further investigation.

Challenges and strategies for clinical trials in propionic and methylmalonic acidemias.

Clinical trial development in rare diseases poses significant study design and methodology challenges, such as disease heterogeneity and appropriate patient selection, identification and selection of key endpoints, decisions on study duration, choice of control groups, selection of appropriate statistical analyses, and patient recruitment. Therapeutic development in organic acidemias (OAs) shares many challenges with other inborn errors of metabolism, such as incomplete understanding of natural history, heterogenous disease presentations, requirement for sensitive outcome measures and difficulties recruiting a small sample of participants. Here, we review strategies for the successful development of a clinical trial to evaluate treatment response in propionic and methylmalonic acidemias. Specifically, we discuss crucial decisions that may significantly impact success of the study, including patient selection, identification and selection of endpoints, determination of the study duration, consideration of control groups including natural history controls, and selection of appropriate statistical analyses. The significant challenges associated with designing a clinical trial in rare disease can sometimes be successfully met through strategic engagement with experts in the rare disease, seeking regulatory and biostatistical guidance, and early involvement of patients and families.

Identification and characterization of the largest deletion in the PCCA gene causing severe acute early-onset form of propionic acidemia.

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM_000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in the PCCA gene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877 bp length, "NG_008768.1:g.185211_403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM_000060.4(BTD):c.1330G > C.

Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy.

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

Translational Pharmacokinetic/Pharmacodynamic Model for mRNA-3927, an Investigational Therapeutic for the Treatment of Propionic Acidemia.

Propionic acidemia (PA) is an ultrarare disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC), composed of PCCA and PCCB subunits. An enzyme replacement therapy is being developed using dual messenger RNA (mRNA) therapy composed of lipid nanoparticles (LNPs) encapsulating mRNAs encoding PCCA and PCCB subunits of the PCC enzyme. We herein report on development of a translational semimechanistic pharmacokinetic (PK) and PK/pharmacodynamic (PD) model to quantify the relationship between the mRNA components of mRNA-3927 (an LNP encapsulating PCCA and PCCB mRNAs) and dose levels; PCCA/B mRNA PK and PD responses were assessed as circulating levels of primary disease markers 2-methyl citrate, 3-hydroxypropionate, and propionyl carnitine normalized to acetyl carnitine (C3/C2 ratio) to inform the first-in-human dose range and regimen selection. The translational PK/PD model was developed using preclinical data available in mice with PA, Sprague Dawley rats, and cynomolgus monkeys at dose levels ranging from 0.2 to 9 mg/kg. PCCA/B mRNA PK in mice, rats, and monkeys was adequately described using allometric scaling of volume and clearance parameters. The interspecies preclinical model was scaled allometrically to humans to predict the dose-response relationship in adult and pediatric patients with PA to guide selection of dose range and regimen for the Phase 1 clinical trial (ClinicalTrials.gov Identifier NCT04159103).

Neuropathological report of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive inheritable metabolic disease caused by mutations in the propionyl CoA carboxylase gene (PCC) that affects multiple systems of the human body. Here, we report neuropathological findings of a PA patient. The patient was a male infant who presented with increasing lethargy and poor feeding from four days postpartum. He gradually became comatose and died from complications after liver transplantation at three months old. The results of laboratory examination were consistent with PA, and genetic analysis revealed compound heterozygous mutations in the gene for PCC subunit beta: c.838dupC (rs769968548) and c.1127G>T (rs142982097). Brain-restricted autopsy was performed 23 h after his death, and the neuropathological examination revealed distinct astrocytosis, oligodendrocytic loss, neuronal loss, and demyelination across the brainstem, motor cortex, basal ganglia, and thalamus. Spongiosis, vacuolization, and the appearance of Alzheimer type II astrocytes and activated microglia were observed as well. This is the first brain autopsy report of PA with a clear genetic cause.

[Phenotypes and genotypes of 78 patients with propionic acidemia].

Objective: Propionic acidemia is a rare inherited metabolic disorder caused by propionyl CoA carboxylase (PCC) deficiency. This study aims to analyze the clinical characteristics and gene variations of Chinese patients with propionic acidemia, and to explore the correlation between clinical phenotypes and genotypes. Methods: Single-center, retrospective and observational study. Seventy-eight patients of propionic acidemia (46 males and 32 females) from 20 provinces and autonomous regions were admitted from January 2007 to April 2022. Their age of initial diagnosis ranged from 7 days to 15 years. The clinical manifestations, biochemical and metabolic abnormalities, genetic variations, diagnosis, treatment and outcome were studied. Chi-Square test or Mann-Whitney U test were used for statistical analysis. Results: Among 78 cases, 6 (7.7%) were identified by newborn screening; 72 (92.3%) were clinically diagnosed after onset, and the age of onset was 2 hours after birth to 15 years old; 32 cases had early-onset disease and 40 cases had late-onset disease. The initial manifestations included lethargy, hypotonia, vomiting, feeding difficulties, developmental delay, epilepsy, and coma. Among the 74 cases who accepted gene analysis, 35 (47.3%) had PCCA variants and 39 (52.7%) had PCCB variants. A total of 39 PCCA variants and 32 PCCB variants were detected, among which c.2002G>A and c.229C>T in PCCA and c.838dupC and c.1087T>C in PCCB were the most common variants in this cohort. The variants c.1228C>T and c.1283C>T in PCCB may be related to early-onset type. The variants c.838dupC, c.1127G>T and c.1316A>G in PCCB, and c.2002G>A in PCCA may be related to late-onset disease. Six patients detected by newborn screening and treated at asymptomatic stage developed normal. The clinically diagnosed 72 cases had varied complications. 10 (12.8%) cases of them died. 62 patients improved after metabolic therapy by L-carnitine and diet. Six patients received liver transplantation because of recurrent metabolic crisis. Their clinical symptoms were markedly improved. Conclusion: The clinical manifestations of propionic acidemia are complex and lack of specificity. Newborn screening and high-risk screening are keys for early treatment and better outcome. The correlation between the genotype and phenotype of propionic acidemia is unclear, but certain variants may be associated with early-onset or late-onset propionic acidemia.

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.

[Differential diagnosis of a Chinese pedigree with methylmalonic acidemia by next-generation sequencing].

OBJECTIVE: To explore the genetic etiology of a child with suspected propionic acidemia. METHODS: Genomic DNA was extracted from peripheral blood sample of the child and subjected to high-throughput sequencing to screen pathogenic variants of genes associated with methylmalonic acidemia and propionic acidemia, including MUT, MMACHC, MMAA, MMAB, MMADHC, LMBRD1, PCCA, PCCB and SLC22A5. Candidate variants were verified by Sanger sequencing of the proband, her parents and sister. RESULTS: The proband was found to harbor two pathogenic variants of the MUT gene, namely c.1560+2T>C and c.729_730insTT (p.Asp244fs), but not in genes associated with propionic acidemia. Her sister and father had carried c.1560+2T>C, and her mother had carried c.729_730insTT (p.Asp244fs). CONCLUSION: The proband was diagnosed as methylmalonic acidemia due to compound heterozygous variants of c.1560+2T>C and c.729_730insTT (p.Asp244fs) of the MUT gene. Her elder sister and parents were all carriers. Genetic testing has facilitated differential diagnosis of methylmalonic acidemia and propionic acidemia in this pedigree.

Analysis of the relationship between phenotypes and genotypes in 60 Chinese patients with propionic acidemia: a fourteen-year experience at a tertiary hospital.

BACKGROUND: Propionic acidemia is a severe inherited metabolic disorder, caused by the deficiency of propionyl-CoA carboxylase which encoded by the PCCA and PCCB genes. The aim of the study was to investigate the clinical features and outcomes, molecular epidemiology and phenotype-genotype relationship in Chinese population. METHODS: We conducted a retrospective study of 60 Chinese patients diagnosed at Peking University First Hospital from 2007 to 2020. Their clinical and laboratory data were reviewed. The next-generation sequencing was conducted on blood samples from 58 patients. RESULTS: Only 5 (8.3%) patients were identified by newborn screening. In the rest 55 patients, 25 had early-onset (≤ 3 months) disease and 30 had late-onset (> 3 months) disease. Neurological abnormalities were the most frequent complications. Five cases detected by newborn screening had basically normal development. Nine (15%) cases died in our cohort. 24 patients (41.4%) harbored PCCA variants, and 34 (58.6%) harbored PCCB variants. 30 (11 reported and 19 novel) variants in PCCA and 28 (18 reported and 10 novel) variants in PCCB mere identified. c.2002G>A and c.937C>T in PCCA, and c.838dupC in PCCB were the most common variants in this cohort, with the frequency of 13.9% (6/44 alleles), 13.9% (6/44 alleles) and 12.5% (8/64 alleles), respectively. There was no difference in clinical features and outcomes between patients with PCCA and PCCB variants. Certain variants with high frequencies and homozygotes may be associated with early-onset or late-onset propionic acidemia. CONCLUSIONS: Although the genotype-phenotype correlation is still unclear, certain variants seemed to be related to early-onset or late-onset propionic acidemia. Our study further delineated the complex clinical manifestations of propionic acidemia and expanded the spectrum of gene variants associated with propionic acidemia.