Engineered leucine decarboxylases
Inventors
Asfaha, Jonathan Benjamin • Das, Subhamoy • Du, Faye Loan • Jenne, Stephan • Kruse, Nikki D. • Liu, Joyce • McCluskie, Kerryn • Mehmood, Roasa • Silverman, Adam P. • Vallieu, Kristen Jean
Assignees
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Publication Number
US-12502421-B2
Publication Date
2025-12-23
Expiration Date
Abstract
The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase polypeptides. In some embodiments, the engineered leucine decarboxylase polypeptides display enhanced catalytic activity, as well as reduced sensitivity to proteolysis, and/or increased tolerance to low pH environments. In some embodiments, the engineered leucine decarboxylase polypeptides are optimized to provide improved storage stability. The present invention also provides methods for the use of the compositions comprising the engineered leucine decarboxylase polypeptides for therapeutic and industrial purposes.
Core Innovation
The invention relates to engineered leucine decarboxylase polypeptides comprising an amino acid sequence having at least 96% sequence identity to a reference sequence corresponding to SEQ ID NO: 828. The engineered polypeptides comprise at least one substitution at amino acid positions defined relative to the reference sequence corresponding to SEQ ID NO: 828, including 202H, 272A, 290I, or 383S, or combinations thereof.
The engineered leucine decarboxylase polypeptides show increased leucine decarboxylase activity in a gastric environment compared to a reference leucine decarboxylase defined by SEQ ID NO: 828. The invention further asserts improved thermostability, increased acid tolerance, reduced sensitivity to proteolysis, improved storage stability, and reduced immunogenicity and serum stability compared to wild-type Planctomycetaceae LDC.
The invention also encompasses uses of the engineered leucine decarboxylase polypeptides for reducing plasma levels of leucine, isoleucine, alloisoleucine, and/or ketoisocaproic acid in a subject. These uses are directed to treating and preventing symptoms of metabolic diseases associated with elevated branched-chain amino acid and metabolite levels, including maple syrup urine disease, isovaleric acidemia, and 3-methylcrotonyl-CoA carboxylase deficiency.
Claims Coverage
The independent claims include an engineered leucine decarboxylase polypeptide defined by at least 96% sequence identity to SEQ ID NO: 828 and at least one specified substitution, together with a functional feature of increased activity in a gastric environment. The claim coverage also includes therapeutic methods for reducing specific plasma metabolites and treating named metabolic diseases.
Engineered leucine decarboxylase sequence identity with specified substitution positions
An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 96% sequence identity to a reference sequence corresponding to SEQ ID NO: 828, wherein the amino acid sequence comprises at least one substitution according to 202H, 272A, 290I, or 383S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
Increased leucine decarboxylase activity in a gastric environment
An engineered leucine decarboxylase polypeptide that shows increased leucine decarboxylase activity in a gastric environment compared to a reference leucine decarboxylase defined by SEQ ID NO: 828.
Reduction of plasma branched-chain amino acid and related metabolite levels by administration of an engineered LDC
A method for reducing plasma levels of leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid in a subject by administering an effective amount of an engineered leucine decarboxylase.
Treatment directed to maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency
The method where the disease or condition linked to elevated plasma leucine, isoleucine, alloisoleucine, and/or ketoisocaproic acid levels is maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.
Overall, the claim coverage focuses on engineered leucine decarboxylase polypeptides defined by high sequence identity to SEQ ID NO: 828 and specified substitution positions, with functional refinement for increased activity in a gastric environment, and therapeutic methods directed to lowering specific plasma amino acids and metabolites and treating named metabolic diseases.
Stated Advantages
Higher leucine decarboxylase activity under gastric-environment conditions compared to a reference leucine decarboxylase defined by SEQ ID NO: 828.
Improved thermostability.
Increased acid tolerance.
Resistance to proteolysis.
Reduced immunogenicity and serum stability compared to wild-type Planctomycetaceae LDC.
Improved storage stability.
Suppression of plasma leucine and related metabolites such as KIC in iMSUD mouse models and healthy cynomolgus monkeys.
Documented Applications
Treating maple syrup urine disease (MSUD) by reducing plasma leucine, isoleucine, alloisoleucine, and/or ketoisocaproic acid levels through administration of an engineered leucine decarboxylase.
Treating isovaleric acidemia by reducing plasma leucine, isoleucine, alloisoleucine, and/or ketoisocaproic acid levels through administration of an engineered leucine decarboxylase.
Treating 3-methylcrotonyl-CoA carboxylase deficiency by reducing plasma leucine, isoleucine, alloisoleucine, and/or ketoisocaproic acid levels through administration of an engineered leucine decarboxylase.
Therapeutic and prophylactic use for treating and preventing symptoms of metabolic diseases associated with elevated branched-chain amino acid and metabolite levels.
Pharmacodynamic study use showing dose-dependent suppression of plasma leucine, KIC, and methionine in iMSUD mouse models and healthy cynomolgus monkeys.
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