Engineered leucine decarboxylases
Inventors
Liu, Joyce • Teadt, Leann Quertinmont • Dellas, Nikki • Jenne, Stephan • Du, Faye Loan • Vallieu, Kristen Jean • McCluskie, Kerryn
Assignees
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Publication Number
US-12241100-B2
Publication Date
2025-03-04
Expiration Date
Abstract
The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase (LDC) polypeptides. In some embodiments, the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis, and/or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability. The present invention also provides methods for the use of the compositions comprising the engineered LDC polypeptides for therapeutic and industrial purposes.
Core Innovation
The invention relates to engineered leucine decarboxylase (LDC) polypeptides, including engineered variants, fragments, and analogs, and to isolated polypeptides and polypeptide compositions comprising such engineered LDCs. The engineered LDC polypeptides are defined by amino acid sequence identity to the entire sequence set forth in SEQ ID NO: 12, with the amino acid positions numbered with reference to SEQ ID NO: 12.
The engineered LDC polypeptides include a substitution at position 364 selected from L364K and L364R, together with sequence identity criteria relative to SEQ ID NO: 12. The disclosed engineered LDCs are directed to enhanced catalytic activity and improved enzyme properties as compared to reference LDCs defined by SEQ ID NO: 12, including reduced sensitivity to proteolysis, improved low-pH tolerance, and greater thermostability.
The disclosure further characterizes the engineered LDC polypeptides as having improved storage stability and additional attributes, including reduced immunogenicity and serum stability. The document also describes polynucleotides encoding the engineered LDC polypeptides, expression constructs and host cells for producing engineered LDCs, and compositions that incorporate the engineered LDC polypeptides.
Claims Coverage
The claim coverage centers on engineered leucine decarboxylase polypeptides defined by high sequence identity to SEQ ID NO: 12 together with a position 364 substitution selected from L364K and L364R. Dependent claims further require improved activity and stability properties relative to SEQ ID NO: 12, and extend coverage to isolation from a recombinant expression host and to compositions that include the engineered polypeptide.
Engineered leucine decarboxylase sequence identity to SEQ ID NO: 12
An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the entire sequence set forth in SEQ ID NO: 12.
Position 364 substitution selected from L364K and L364R
The amino acid sequence comprises a substitution at position 364, selected from L364K and L364R, wherein the amino acid positions are numbered with reference to the amino acid sequence of SEQ ID NO: 12.
Greater leucine activity than SEQ ID NO: 12
The engineered leucine decarboxylase polypeptide has greater activity on leucine than the leucine decarboxylase polypeptide in SEQ ID NO: 12.
More resistant to proteolysis than SEQ ID NO: 12
The engineered leucine decarboxylase polypeptide is more resistant to proteolysis than the leucine decarboxylase polypeptide in SEQ ID NO: 12.
More thermostable than SEQ ID NO: 12
The engineered leucine decarboxylase polypeptide is more thermostable than the leucine decarboxylase polypeptide in SEQ ID NO: 12.
Isolated from a recombinant expression host
The engineered leucine decarboxylase polypeptide is isolated from a recombinant expression host.
Composition comprising the engineered polypeptide
A composition includes at least one engineered leucine decarboxylase polypeptide as specified in claim 1.
Overall, the claims cover engineered leucine decarboxylase polypeptides defined by high sequence identity to SEQ ID NO: 12 and an L364K or L364R substitution at position 364. The inventive features further include greater leucine activity, increased proteolysis resistance, greater thermostability, isolation from a recombinant expression host, and compositions comprising the engineered polypeptide.
Stated Advantages
Enhanced catalytic activity.
Reduced sensitivity to proteolysis.
Increased low-pH tolerance.
Improved storage stability.
Reduced immunogenicity.
Serum stability.
Documented Applications
Pharmacodynamic suppression of plasma leucine after dosing in healthy cynomolgus monkeys.
Pharmacodynamic suppression of plasma leucine in iMSUD mouse models, with no effect on certain other amino acids.
Treating maple syrup urine disease (MSUD) and leucineuria, including contexts involving BCKD complex deficiency.
Industrial and therapeutic applications.
Pharmaceutical/gene therapy formulations including compositions incorporating the engineered LDC polypeptides.
mRNA therapy formulations including compositions incorporating the engineered LDC polypeptides.
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