Synthetic methylmalonyl-CoA mutase transgene for the treatment of MUT class methylmalonic acidemia (MMA)
Inventors
Venditti, Charles P. • CHANDLER, Randy J.
Assignees
US Department of Health and Human Services
Publication Number
US-10307469-B2
Publication Date
2019-06-04
Expiration Date
2034-03-14
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Abstract
Synthetic polynucleotides encoding human methylmalonyl-CoA mutase (synMUT) and exhibiting augmented expression in cell culture and/or in a subject are described herein. An adeno-associated viral (AAV) gene therapy vector encoding synMUT under the control of a liver-specific promoter (AAV2/8-HCR-hAAT-synMUT-RBG) successfully rescued the neonatal lethal phenotype displayed by methylmalonyl-CoA mutase-deficient mice, lowered circulating methylmalonic acid levels in the treated animals, and resulted in prolonged hepatic expression of the product of synMUT transgene in vivo, human methylmalonyl-CoA mutase (MUT).
Core Innovation
The invention provides synthetic polynucleotides encoding a human methylmalonyl-CoA mutase gene (synMUT) that are codon-optimized to enhance expression in eukaryotic cells. These synthetic sequences exhibit augmented expression in cell culture and in subjects, improving the production of the methylmalonyl-CoA mutase enzyme relative to naturally occurring human MUT gene sequences. The augmented expression is achieved by replacing less commonly used codons with more commonly used ones, optimizing transcriptional and translational efficiency, and eliminating cryptic splicing and RNA instability motifs.
Methylmalonic acidemia (MMA) is an autosomal recessive disorder caused by defects in the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), which catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA, a critical step in metabolizing certain amino acids and fatty acids. MMA patients accumulate methylmalonic acid, leading to metabolic instability, seizures, strokes, kidney failure, and potentially death. Current treatments are limited to dietary restrictions, which are inadequate to prevent disease progression and metabolic crises.
The synthetic MUT transgene can be delivered via viral or non-viral vectors as a therapeutic to restore MUT function in MMA patients, preventing metabolic instability and ameliorating disease progression. Additionally, synMUT can be used to produce MUT enzyme for enzyme replacement therapy through various routes such as oral or intravenous administration. The invention also covers expression vectors, transgenic animals, gene editing methods, and delivery systems to enable the therapeutic use of synMUT, demonstrating efficacy in preclinical models including rescue of neonatal lethality and reduction of disease biomarkers.
Claims Coverage
The patent includes one independent claim focused on a method of treating diseases mediated by methylmalonyl-CoA mutase using synthetic polynucleotides.
Use of synthetic codon-optimized methylmalonyl-CoA mutase polynucleotides for therapy
Administering to a subject a therapeutic amount of a methylmalonyl-CoA mutase produced from a synthetic polynucleotide selected from sequences comprising SEQ ID NO:1 or codon-optimized polynucleotides having at least about 80% identity to SEQ ID NO:1 encoding the polypeptide of SEQ ID NO:2, excluding naturally occurring sequence SEQ ID NO:3.
Application to methylmalonic acidemia
Specifying that the disease or condition treated is methylmalonic acidemia (MMA).
Polynucleotide identity thresholds
Use of synthetic polynucleotides with at least about 90%, 95%, 97%, or 99% identity to SEQ ID NO:1 to achieve therapeutic effect.
Increased expression of synthetic polynucleotide
Using synthetic methylmalonyl-CoA mutase polynucleotides exhibiting increased expression in a host relative to naturally occurring human sequence SEQ ID NO:3, achieved by codon optimization and replacement of less commonly used codons.
Formulation and administration
Formulating the synthetic methylmalonyl-CoA mutase with a pharmaceutically acceptable carrier and administering either the enzyme or expression vector encoding the synthetic polynucleotide to the subject.
The claims cover therapeutic methods employing synthetic codon-optimized methylmalonyl-CoA mutase polynucleotides with enhanced expression relative to natural sequences, specifically detailing identity thresholds, application to MMA, and modes of formulation and administration.
Stated Advantages
The synthetic polynucleotides exhibit augmented expression of methylmalonyl-CoA mutase relative to naturally occurring sequences, enabling increased enzyme production.
Gene therapy using synMUT rescues neonatal lethality in mouse models of MMA, improves growth, and lowers circulating methylmalonic acid levels.
The AAV8-hAAT-synMUT vector demonstrated a better safety profile with less genotoxicity and lower incidence of hepatocellular carcinoma compared to other AAV constructs.
Documented Applications
Treatment of methylmalonic acidemia (MMA) in subjects via administration of synMUT gene therapy or enzyme replacement therapy.
Use of synMUT for in vitro production of methylmalonyl-CoA mutase enzyme for enzyme replacement therapies.
Gene delivery using viral vectors, including adeno-associated virus vectors, for restoration of enzyme function in MMA animal models and potentially humans.
Generation of transgenic animals carrying synMUT polynucleotides for recombinant protein production and disease modeling.
Genome editing approaches using synMUT sequences in combination with engineered nucleases (e.g., ZFNs, TALENs, CRISPR/Cas) for correcting MUT deficiency ex vivo or in vivo.
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