US2023295599A1PendingUtilityA1

Engineered phenylalanine ammonia lyase polypeptides

78
Assignee: CODEXIS INCPriority: Feb 13, 2017Filed: Jan 13, 2023Published: Sep 21, 2023
Est. expiryFeb 13, 2037(~10.6 yrs left)· nominal 20-yr term from priority
Inventors:Chinping ChngWilliam Casey HallowsNicholas J. AgardOscar AlvizoNikki DellasGjalt W. HuismanJohn Joseph Nicols
C12N 9/88A61K 9/0053C12Y 403/01024A61K 38/00A61K 9/0019A61P 3/00A61P 43/00A61K 48/00C12N 15/00
78
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

The present invention provides engineered phenylalanine ammonia lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia lyase (PAL) polypeptides. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis and increased tolerance to storage at elevated temperatures. In some embodiments, the engineered PAL polypeptides contain fewer phenylalanine residues than wild-type PAL polypeptides. The present invention also provides methods for the use of the compositions comprising the engineered PAL polypeptides for therapeutic and industrial purposes.

Claims

exact text as granted — not AI-modified
We claim: 
     
         1 . An engineered polypeptide comprising an amino acid sequence having at least 85% sequence identity to at least one of SEQ ID NO: 4, 10, and/or 12, wherein the amino acid positions of said amino acid sequences are numbered with reference to the amino acid sequence of SEQ ID NO:2. 
     
     
         2 . The engineered polypeptide of  claim 1 , wherein the engineered polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 54/56/162/204/285, 54/56/162/204/398, 54/56/204/285, 54/56/204/398, 54/56/204/398/472, 54/56/285, 54/56/398, 54/56/398/472, 54/162/204/398, 54/162/398, 54/204/285/398/472, 54/204/398, 54/285/398, 54/285/398/472, 56/162/398, 56/204/285, 56/204/398, 56/204/398/460, 56/204/398/472, 56/285, 56/285/398/472, 56/398, 56/398/472, and 201/204/398, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:12. 
     
     
         3 . The engineered polypeptide of  claim 1 , wherein the engineered polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 16, 18, 39, 47, 54, 59, 73, 91, 209, 214, 285, 290, 305, 307, 364, 407, 450, 470, 503, 521, 524, and 565, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:12. 
     
     
         4 . The engineered polypeptide of  claim 1 , wherein the engineered polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 15/18/39/47/54/59/73/91/209/214/285/290/307/364/407/450/470/503/521/524/565, 16/18/19/214/407, 16/18/23/47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/503/521/524/565, 16/18/39/43/44/59/209/503/565, 16/18/39/45/47/54/59/73/98/209/214/285/290/305/364/407/450/470/503/521/524/565, 16/18/39/47/49/54/59/73/209/285/290/305/307/364/450/470/503/521/524/565, 16/18/39/47/54/59/73/91/98/285/290/305/307/364/450/470/503/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/305/307/313/364/450/503/521/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/503/521/524, 16/18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/503/521/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/521/524/565, 16/18/39/47/54/59/73/91/209/214/285/290/305/307/364/407/450/503/521/524, 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39/54/59/73/91/285/290/307/364/407/450/503/524, 39/54/59/73/98/209/214/285/290/305/307/364/450/470/503, 39/54/59/73/209/214/285/290/305/307/364/407/450/503/521/565, 39/54/59/73/290/305/307/364/407/470/503/521/524/565, 39/54/59/91/98/209/287/290/291/307/364/407/450/470/521/524, 39/54/59/91/98/285/290/503/521/524/565, 39/54/59/91/209/214/285/290/305/307/364/407/470/503/524/565, 39/54/73/91/209/214/285/290/305/364/503/524/565, 39/59/73/209/285/290/305/307/364/407/450/470/503/521/524/565, 39/59/202/407/524/565, 39/59/364/450/470/565, 39/73/290/307/364/407/450/524/565, 39/209/214/407/450/470/503/521, 39/209/450/470/503, 39/214/285/290/305/307/364/503/521/524, 39/214/307/503/524, 47, 47/54/59/73/91/209/214/285/290/305/307/364/407/450/470/503/521/524/565, 47/54/59/73/91/209/285/290/305/307/364/407/450/470/503/521/524, 47/54/59/73/91/209/285/290/305/307/364/407/450/503/524/565, 47/54/59/73/91/285/290/311/364/407/503/521/524/565, 47/54/59/73/91/285/305/307/407/470/521/524, 47/54/59/73/98/209/214/285/290/305/307/364/407/450/470/503/521/524, 47/54/59/73/209/285/290/364/407/432/450/470/503/521/524/565, 47/54/73/91/209/214/285/290/305/307/364/407/450/470/503/521/524/565, 47/54/73/91/285/290/307/364/450/470/503/521/524/565, 47/54/91/97/209/364/503/565, 47/55, 47/59/91/407/470/503/565, 47/59/91/503/521, 47/59/209, 47/59/450/470/503, 47/73, 47/73/91/214, 47/73/91/285/290/305/307/364/450/470/503/521/524, 47/73/91/290/407/416/450/503/521/524, 47/73/209/364/450/521, 47/73/214/305/307/364/503/565, 47/73/214/305/407/503/565, 47/73/214/364/407, 47/73/407/470/503, 47/209/214/305/307/364/503/521/524, 47/209/503/524/565, 47/214/364/503, 47/214/407, 47/285/290/364/503/565, 47/305, 47/364/407/470/521/524, 47/364/524/565, 47/407, 47/407/450/521/524, 47/407/565, 47/524/565, 51/407/470/503/524, 54/59/73/91/214/285/290/364/407/470/503/524/565, 54/59/91/209/407, 54/73/91/214/450/503/521/524, 54/73/91/305/364/407/450/470/503/521, 54/73/450, 54/209/285/290/305/307/364/407/450/470/503/565, 54/209/407/450/470/565, 54/209/470/503/524/565, 54/450, 54/503, 55/290/305/364/450/503, 59/73/290/407/503/554, 59/73/305/307/503, 59/91/407/450, 59/209/364/470/503, 59/214, 59/214/364/407/452/503, 59/214/450/503, 59/407/503/521/524, 59/450/503, 59/565, 73/91/214/524/565, 73/91/521, 91/97/209/285, 91/209/214/255/285/290/307/450, 91/209/214/364/407/450/470/565, 91/209/214/407/503/565, 91/209/214/450/521/524/565, 91/209/214/470/565, 91/209/407, 91/214, 209, 209/214/305, 209/214/307/407/450/503, 209/214/407/450/470/503, 209/470, 214/285/307/503/565, 214/407/503/565, 214/470/503, 214/470/521, 290/470, 305/407/521, 364, 364/407/419/450/470/521/565, 364/407/565, 364/470/503/521/524, 407, 407/450/565, 407/503, 450/503, 450/524, 503, and 524, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:2. 
     
     
         5 . An engineered phenylalanine ammonia lyase polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, 10, and/or 12, or a functional fragment thereof. 
     
     
         6 . An engineered phenylalanine ammonia lyase polypeptide, wherein said engineered phenylalanine ammonia lyase polypeptide comprises an engineered polypeptide provided in  claim 1 . 
     
     
         7 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered phenylalanine ammonia lyase polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 4, 10, and/or 12, or a functional fragment thereof. 
     
     
         8 . The engineered phenylalanine ammonia lyase polypeptide of  claim 7 , wherein said engineered polypeptide is an  Anabaena variabilis  variant enzyme. 
     
     
         9 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide is more thermostable than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         11 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide more resistant to proteolysis than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         12 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered phenylalanine ammonia lyase polypeptide is resistant to proteolysis by at least one digestive tract enzyme, than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         13 . The engineered phenylalanine ammonia lyase polypeptide of  claim 12 , wherein said engineered phenylalanine ammonia lyase polypeptide is more resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         14 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide is more acid stable than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         15 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said polypeptide is purified. 
     
     
         16 . An engineered polynucleotide sequence encoding at least one engineered polypeptide of  claim 1 . 
     
     
         17 . The engineered polynucleotide sequence of  claim 16 , wherein said polynucleotide is codon-optimized. 
     
     
         18 . An expression vector comprising at least one engineered polynucleotide sequence of  claim 16 . 
     
     
         19 . An expression vector comprising at least one engineered polynucleotide sequence of  claim 17 . 
     
     
         20 . The expression vector of  claim 19 , further comprising at least one control sequence. 
     
     
         21 . The expression vector of  claim 20 , wherein said control sequence comprises a promoter. 
     
     
         22 . A host cell transformed with at least one expression vector of  claim 18 . 
     
     
         23 . A method of producing an engineered phenylalanine ammonia lyase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered polypeptide and/or phenylalanine ammonia lyase polypeptide of  claim 1 , under suitable culture conditions, such that at least one engineered phenylalanine ammonia lyase polypeptide is produced. 
     
     
         24 . The method of  claim 23 , further comprising recovering at least one engineered phenylalanine ammonia lyase polypeptide from the culture and/or host cells. 
     
     
         25 . The method of  claim 23 , further comprising the step of purifying said at least one engineered phenylalanine ammonia lyase polypeptide. 
     
     
         26 . An engineered phenylalanine ammonia lyase polypeptide produced according to the method of  claim 23 . 
     
     
         27 . A composition comprising at least one engineered phenylalanine ammonia lyase polypeptide of  claim 1 . 
     
     
         28 . A composition comprising at least one engineered polynucleotide of  claim 16 . 
     
     
         29 . The composition of  claim 27 , wherein said composition is a pharmaceutical composition. 
     
     
         30 . The composition of  claim 29 , further comprising at least one pharmaceutically acceptable excipient and/or carrier. 
     
     
         31 . The composition of  claim 29 , wherein said composition is suitable for the treatment of phenylketonuria. 
     
     
         32 . The composition of  claim 29 , wherein said composition is suitable for the treatment of elevated blood phenylalanine. 
     
     
         33 . The composition of  claim 29 , wherein said composition is suitable for the treatment of tyrosinemia. 
     
     
         34 . The composition of  claim 29 , wherein said composition is suitable for the treatment of elevated blood tyrosine. 
     
     
         35 . The composition of  claim 28 , wherein said composition is suitable for use in gene therapy. 
     
     
         36 . The composition of  claim 35 , wherein said composition is suitable for use in gene therapy to treat phenylketonuria, elevated blood phenylalanine, tyrosinemia, and/or elevated blood tyrosinemia. 
     
     
         37 . The composition of  claim 28 , wherein said composition is suitable for use in mRNA therapy. 
     
     
         38 . The composition of  claim 37 , wherein said composition is suitable for use in mRNA therapy to treat phenylketonuria, elevated blood phenylalanine, tyrosinemia, and/or elevated blood tyrosinemia. 
     
     
         39 . The composition of  claim 29 , wherein said composition is suitable for oral administration to a human. 
     
     
         40 . The composition of  claim 39 , wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. 
     
     
         41 . The composition of  claim 40 , wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating. 
     
     
         42 . The composition of  claim 29 , wherein said composition is suitable for parenteral injection into a human. 
     
     
         43 . The composition of  claim 29 , wherein said composition is coadministered with at least one additional therapeutically effective compound. 
     
     
         44 . The composition of  claim 29 , wherein said composition comprises at least one additional therapeutically effective compound. 
     
     
         45 . A method for treating and/or preventing the symptoms of phenylketonuria in a subject, comprising providing a subject having phenylketonuria, and providing the composition of  claim 29 , to said subject. 
     
     
         46 . The method of  claim 45 , wherein the phenylalanine concentration in the blood of said subject is reduced upon providing said composition to said subject. 
     
     
         47 . The method of  claim 45 , wherein the cinnamic acid concentration in the blood of said subject is increased upon providing said composition to said subject. 
     
     
         48 . The method of  claim 45 , wherein said symptoms of phenylketonuria are ameliorated. 
     
     
         49 . A method for treating and/or preventing the symptoms of phenylketonuria in a subject, comprising providing a subject having phenylketonuria, and providing the composition of  claim 29 , wherein said subject is able to eat a diet that is less restricted in its phenylalanine content than diets required by subjects who have not been provided said composition. 
     
     
         50 . The method of  claim 49 , wherein said subject is an infant, child, young adult, or adult.

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