Treatment Of Methylmalonic Acidemia Research Articles

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.

Treatment of metabolic disorders using genomic technologies: Lessons from methylmalonic acidemia.

Hereditary methylmalonic acidemia (MMA) caused by deficiency of the enzyme methylmalonyl-CoA mutase (MMUT) is a relatively common and severe organic acidemia. The recalcitrant nature of the condition to conventional dietary and medical management has led to the use of elective liver and combined liver-kidney transplantation in some patients. However, liver transplantation is intrinsically limited by organ availability, the risks of surgery, procedural and life-long management costs, transplant comorbidities, and a remaining underlying risk of complications related to MMA despite transplantation. Here, we review pre-clinical studies that present alternative approaches to solid organ transplantation as a treatment for MMUT MMA, including adeno-associated viral gene addition therapy, mRNA therapy, and genome editing, with and without nuclease enhancement.

Identification of 2,2-Dimethylbutanoic Acid (HST5040), a Clinical Development Candidate for the Treatment of Propionic Acidemia and Methylmalonic Acidemia.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.

Clinical value of liver transplantation in the treatment of methylmalonic acidemia

Methylmalonic acidemia (MMA) is a rare autosomal recessive disorder, with the manifestation of recurrent and severe episodes of acute metabolic decompensation or a variety of long-term complications that require timely treatment.As liver transplantation (LT) developing widely, it has become an effective treatment for patients with severe MCM deficiency.Although LT does not solve the fundamental problem of gene defect, it can prolong survival, improve the quality of life of patients and reduce recurrent hospitalization rate, as well as lower urine, plasma MMA and propionyl carnitine levels and prevent metabolic stroke.After LT, the metabolism-correcting medications are still partially needed.Neurological symptoms of some patients can be alleviated but renal damage may remain irreversible.The domestic liver transplantation in the treatment of methylmalonic acidemia is being carried out, but there is still a lack of research on the effect of LT on MMA.The paper is to make a review of the clinical value of liver transplantation in the treatment of methylmalonic acidemia in recent years in hope of sharing the information on this issue with more people. Key words: Methylmalonic acidemia; Methylmalonic acid; Liver transplantation

Pathophysiology of propionic and methylmalonic acidemias. Part 2: Treatment strategies.

Despite realizing increased survival rates for propionic acidemia (PA) and methylmalonic acidemia (MMA) patients, the current therapeutic regimen is inadequate for preventing or treating the devastating complications that still can occur. The elucidation of pathophysiology of these complications allows us to evaluate and rethink treatment strategies. In this review we display and discuss potential therapy targets and we give a systematic overview on current, experimental and unexplored treatment strategies in order to provide insight in what we have to offer PA and MMA patients, now and in the future. Evidence on the effectiveness of treatment strategies is often scarce, since none were tested in randomized clinical trials. This raises concerns, since even the current consensus on best practice treatment for PA and MMA is not without controversy. To attain substantial improvements in overall outcome, gene, mRNA or enzyme replacement therapy is most promising since permanent reduction of toxic metabolites allows for a less strict therapeutic regime. Hereby, both mitochondrial-associated and therapy induced complications can theoretically be prevented. However, the road from bench to bedside is long, as it is challenging to design a drug that is delivered to the mitochondria of all tissues that require enzymatic activity, including the brain, without inducing any off-target effects. To improve survival rate and quality of life of PA and MMA patients, there is a need for systematic (re-)evaluation of accepted and potential treatment strategies, so that we can better determine who will benefit when and how from which treatment strategy.

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.

48. Treatment of Methylmalonic Acidemia by Promoterless Gene-Targeting Using Adeno-Associated Viral (AAV) Mediated Homologous Recombination

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism most typically caused by mutations in methylmalonyl-CoA mutase (MUT). While the hallmark of this disease is elevated levels of methylmalonic acid in the plasma, other disease related metabolites such as methylcitrate are elevated in the plasma as well. Patients with MMA suffer from frequent and potential lethal bouts of metabolic instability that can be eliminated by liver transplantation. Adeno-associated viral (AAV) gene therapy has shown great promise as the treatment for MMA in a murine model of the disease. However, a majority of the AAV-treated mice developed hepatocellular cancer, which was determined to arise from AAV-mediated insertional mutagenesis. In an attempt to create a safer gene therapy platform for the treatment of MMA, we created a novel vector for site-specific gene addition of human MUT into the mouse albumin (Alb) locus. This promoterless AAV vector contains a 2A-peptide coding sequence proximal to a codon-optimized human MUT gene. The 2A-MUT sequence is flanked by arms of homology immediately upstream of mouse Alb stop codon. Since albumin is expressed exclusively in hepatocytes, we prepared an AAV serotype 8 vector to take advantage of the murine liver tropism conferred by this capsid. This newly created vector was named AAV8-Alb-A2-MUT. After site-specific integration of the vector into the Alb locus in the liver, ribosomal skipping generates both Alb and MUT as separate proteins derived from the same transcript. We delivered a dose of 2.5e12 GC of AAV8-Alb-2A-MUT to five mice with MMA by intraperitoneal injection at birth. At one month post-injection, we observed increased hepatic expression of the MUT by western blot, improved growth and a significant reduction of disease related metabolites, in the treated MMA mice (Table 1Table 1). This gene delivery approach is anticipated to provide permanent hepatic transgene expression while reducing the risk of off-target integration and vector-mediated insertional mutagenesis.Table 1Markers of Correction in MMA Murine ModelUntreated (n=3-8)AAV8-Alb-A2-MUT (n=5)P ValuePercent of Wild-type Mut ExpressionND11.3+/-9.1NAPlasma Methylmalonic Acid (uM)969+/−396563+/−98<0.05Plasma Methylcitrate (nM)5, 020+/−8732, 904+/−638<0.0001Weight at 1 Month6.9+/−1.610.7+/−0.6<0.003 View Table in HTML

353. 1-13C-Propionate Oxidation as a Measure of Methylmalonyl-CoA Mutase (MUT) Activity in Methylmalonic Acidemia

Isolated methylmalonic acidemia (MMA) is a grave autosomal recessive inborn error of metabolism, caused by a defect in methylmalonyl-CoA mutase (MUT). In some severe patients, liver (LT), kidney (KT), or combined liver and kidney (LKT) transplantation has been used as a surgical treatment for MMA. Previous studies have shown that 1-13C-propionate oxidation can be used to measure whole body MUT activity in gene therapy treated and transgenic MMA mice and have encouraged translation to patients, with the aim of using this approach as an outcome measure in future gene therapy clinical trials.We therefore investigated 1-13C-propionate oxidation in 29 patients with isolated MMA (mut0 N=17, mut− N=6, cblA N=5, cblB N=1, age range: 4-41 years), including 3 KT, 1 LT, and 4 LKT recipients, as well as one patient who received a heterotopic liver allograft post KT (hLKT). In addition, 7 healthy volunteers (3 males, 4 females, age range: 21-44 years) and 8 heterozygote controls (age range:35-62 years) were tested to establish test reproducibility. Baseline CO2 production of the participants was measured using an indirect calorimetry cart before sodium 1-13C-propionate was administered as an oral or G-tube bolus, and breath samples were collected serially over 2 hours via disposable breath collection kits to measure 13CO2 enrichment and subsequently propionate oxidation.Propionate oxidation was decreased in all non-transplanted MMA patients compared with controls (p<0.0001). Both LT and LKT recipients demonstrated complete restoration of oxidation rates to control levels (p=NS), while only minimal oxidation was observed in the single hLKT recipient. KT recipients responsive to vitamin B-12 supplementation (N=2) with cblA MMA showed oxidation rates similar to controls. However, the remaining KT recipient, a severely affected mut0 patient carrying two stop mutations, showed almost no metabolism of label.Repeat testing conducted both on healthy volunteers in triplicate and 7 MMA patients (6 mut, 1 cblA) in duplicate supported excellent reproducibility. Notably, similar oxidation rates were observed in 2 patients despite markedly different plasma methylmalonic acid concentrations between one assessment and the next (1741 vs. 2246 and 2260 vs. 719 µmol/L). This suggests that measured 1-13C-propionate oxidation may be a more consistent and reproducible clinical biomarker than plasma metabolites that depend, in part, on protein intake and kidney function.Our results demonstrate that 1-13C-propionate oxidation, measured using breath testing, has great potential as an outcome measure for therapeutic interventions aimed at increasing hepatic MUT activity, such as gene or cell therapy. It is safe, non-radioactive, minimally invasive, well tolerated by very young patients, and can be frequently repeated over the course of treatment to evaluate the magnitude and sustainability of whole body in vivo MUT activity, with every patient's baseline values serving as an individual pre-treatment control. Furthermore, the method is readily applicable to the study of other inborn errors of metabolism affecting propionate oxidation, such as propionic acidemia.

Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA.

Impaired Health-Related Quality of Life in Children and Families Affected by Methylmalonic Acidemia.

An understanding of health related quality of life (HRQoL) in children and families affected by methylmalonic acidemia (MMA) is important in planning counseling and therapeutic intervention. Liver transplantation (LT) is used as a treatment for MMA; however, its risks and benefits continue to be investigated. The purpose of this study was twofold: (1) to measure HRQoL in children and families affected by MMA using the Pediatric Quality of Life Inventory (PedsQL™) parent version, and (2) to assess the impact of LT on HRQoL by comparing LT and non-LT patient scores and free responses. Parents/caregivers reported lower scores on the majority of the PedsQL™ scales as compared to samples of healthy children, children with solid organ transplants for indications other than MMA, and families affected by chronic conditions. Scores for children with MMA were lowest in school and social functioning and scores for families were lowest in worry and activity impairment. There were no significant differences in LT and non-LT patient scores on the PedsQL™ scales. Our results document the negative impact of MMA on HRQoL.

Treatment of Methylmalonic Acidemia by Liver or Combined Liver-Kidney Transplantation

To assess biochemical, surgical, and long-term outcomes of liver (LT) or liver-kidney transplantation (LKT) for severe, early-onset methylmalonic acidemia/acid (MMA). A retrospective chart review (December 1997 to May 2012) of patients with MMA who underwent LT or LKT at Lucile Packard Children's Hospital at Stanford. Fourteen patients underwent LT (n = 6) or LKT (n = 8) at mean age 8.2 years (range 0.8-20.7). Eleven (79%) were diagnosed during the neonatal period, including 6 by newborn screening. All underwent deceased donor transplantation; 12 (86%) received a whole liver graft. Postoperative survival was 100%. At a mean follow-up of 3.25 ± 4.2 years, patient survival was 100%, liver allograft survival 93%, and kidney allograft survival 100%. One patient underwent liver re-transplantation because of hepatic artery thrombosis. After transplantation, there were no episodes of hyperammonemia, acidosis, or metabolic decompensation. The mean serum MMA at the time of transplantation was 1648 ± 1492 μmol/L (normal <0.3, range 99-4420). By 3 days, post-transplantation levels fell on average by 87% (mean 210 ± 154 μmol/L), and at 4 months, they were 83% below pre-transplantation levels (mean 305 ± 108 μmol/L). Developmental delay was present in 12 patients (86%) before transplantation. All patients maintained neurodevelopmental abilities or exhibited improvements in motor skills, learning abilities, and social functioning. LT or LKT for MMA eradicates episodes of hyperammonemia, results in excellent long-term survival, and suggests stabilization of neurocognitive development. Long-term follow-up is underway to evaluate whether patients who undergo early LT need kidney transplantation later in life.

145. Demonstration of Pre-Clinical Efficacy of rAAV8 Gene Transfer as a Treatment for Methylmalonic Acidemia Using a Vector Suitable for Human Clinical Trials

145. Demonstration of Pre-Clinical Efficacy of rAAV8 Gene Transfer as a Treatment for Methylmalonic Acidemia Using a Vector Suitable for Human Clinical Trials

Gene therapy in a murine model of methylmalonic acidemia using rAAV9-mediated gene delivery

Methylmalonic acidemia (MMA), an inherited metabolic disorder caused by deficient activity of methylmalonyl-CoA mutase (MUT), carries a poor prognosis for long-term survival. While administration of a recombinant adeno-associated virus serotype 8 vector (rAAV8) can rescue Mut-/- mice from neonatal lethality and provide sustained phenotypic correction, translation of gene therapy to human subjects will likely require multiple rounds of systemic administration and ideally, the use of a vector that transduces the kidney. To examine the effectiveness of alternative rAAVs in the treatment of MMA, a serotype 9 rAAV expressing the Mut cDNA was constructed and delivered to newborn Mut-/- mice (n=11). rAAV9 gene therapy directed hepatic transgene expression within 24 hours and effectively rescued the Mut-/- mice from lethality, conferred long-term survival, markedly improved metabolism and resulted in striking preservation of renal function and histology. Systemic re-administration of the vector at a dose similar to that used in human clinical trials (2.5×109 GC rAAV9 per gram) to older, treated Mut-/- mice (n=5) lowered circulating metabolites, increased in vivo propionate oxidative capacity and produced transgene expression in the kidney and liver. Our data support the use of an rAAV9 vector in the acute and chronic treatment of MMA, and highlight the renotropism afforded by this novel serotype.

Variable dietary management of methylmalonic acidemia: metabolic and energetic correlations

Isolated methylmalonic acidemia (MMA) is managed by dietary protein restriction and medical food supplementation. Resting energy expenditure (REE) can be depressed in affected individuals for undefined reasons. The objective was to document the spectrum of nutritional approaches used to treat patients with MMA, measure REE, and analyze the dependence of REE on body composition, biochemical, and nutritional variables. Twenty-nine patients with isolated MMA (22 mut, 5 cblA, 2 cblB; 15 males, 14 females; age range: 2-35 y) underwent evaluation. REE was measured with open-circuit calorimetry and compared with predicted values by using age-appropriate equations. Nutritional regimens were as follows: protein restriction with medical food (n = 17 of 29), protein restriction with medical food and supplemental isoleucine or valine (n = 5 of 29), or the use of natural protein alone for dietary needs (n = 7 of 29). Most mut patients had short stature and higher percentage fat mass compared with reference controls. Measured REE decreased to 74 ± 13.6% of predicted (P < 0.001) in the ≤ 18-y group (n = 22) and to 83 ± 11.1% (P = 0.004) in patients aged >18 y (n = 7). Linear regression modeling suggested that age (P = 0.001), creatinine clearance (P = 0.01), and height z score (P = 0.04) accounted for part of the variance of measured REE per kilogram of fat-free mass (model R² = 0.66, P < 0.0001). There is wide variation in the dietary treatment of MMA. Standard predictive equations overestimate REE in this population primarily due to their altered body composition and decreased renal function. Defining actual energy needs will help optimize nutrition and protect individuals from overfeeding. This trial is registered at clinicaltrials.gov as NCT00078078.

Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut−/− mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus–treated Mut−/− mice (n = 27) have survived for ≥1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut−/− mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.

Clinical outcome of long-term management of patients with vitamin B 12-unresponsive methylmalonic acidemia

We performed a retrospective study of all patients with methylmalonic acidemia diagnosed during the past 20 years. Only those patients who were nonresponsive to vitamin B 12 in vivo and in vitro were included. The final study group consisted of 26 patients, of whom 16 had a neonatal (early) onset; in 10 patients the diagnosis was made after 2 months to 2.2 years (late onset). Of the early-onset patients, 14 (87%) died, with a mean survival time of 1.5 years (range, 10 days to 2.5 years), whereas four of the late-onset patients (40%) died (range, 1.2 to 15 years). At present, eight patients are alive; their mean age is 4.6 years (range, 1 to 10 years). In the early 1970s, treatment was based on the principles of treating patients with phenylketonuria: restricting natural protein intake and supplementing essential amino acids, vitamins, and trace elements. After about 1980, nasogastric tube feeding became a mainstay of the therapy, natural protein restriction became stricter, and the use of essential amino acid mixtures diminished. Carnitine was added to the therapy and, in later years, metronidazole. Since these changes were implemented, the number of episodes of metabolic decompensation and hospitalizations has decreased. Mean survival time of the patients, in particular those with early onset, has only slightly improved, partly because of psychosocial problems in many of these families. Almost all the patients, especially those with early onset, had some degree of neurologic impairment and mental retardation, and many patients were at less than 2 SD for weight or height or both. In contrast, the neurologic and mental status of the late-onset patients was frequently normal, and their weight and height were more often within normal limits. Our results show that the treatment of methylmalonic acidemia still poses considerable problems; despite intense medical efforts and familial stress, the prognosis for the early-onset patients is disappointing. The patients with late-onset disease, however, appear to have a fairly good prognosis with the present therapeutic approach. Liver transplantation or possibly genetic therapy might improve our results in the future. (J P EDIATR 1994;125:903-8)