Human-enzyme mediated depletion of homocysteine for treating patients with hyperhomocysteinemia and homocystinuria
Methods and compositions relating to the engineering of an improved protein with homocyst(e)inase enzyme activity are described. For example, there are disclosed modified cystathionine-γ-lyase (CGL) enzymes comprising one or more amino acid substitutions and capable of degrading homocyst(e)ine. Furthermore, provided are compositions and methods for the treatment of homocystinuria or hyperhomocysteinemia with homocyst(e)ine depletion using the disclosed enzymes or nucleic acids.
1. An isolated, modified human cystathionine-γ-lyase (CGL) enzyme comprising the following substitutions relative to a native human CGL amino acid sequence as shown in SEQ ID NO: 1, wherein the modified enzyme has both homocystinase and homocysteinase activity, wherein the modified enzyme comprises a sequence at least 95% identical to SEQ ID NO: 1, said substitutions comprising isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
2. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 1 , wherein the substitutions comprise E59I, S63L, L91M, R119D, K268R, T311G, E339V, and I353S relative to the native human CGL amino acid sequence as shown in SEQ ID NO: 1.
3. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 1 , wherein the modified human CGL enzyme has a sequence according to SEQ ID NO: 37.
4. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 1 , wherein the enzyme is coupled to at least one polyethylene glycol (PEG).
5. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 4 , wherein the enzyme is coupled to the at least one PEG via one or more lysine residues.
6. A therapeutic formulation comprising the isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 1 , in a pharmaceutically acceptable carrier.
7. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 2 , wherein the enzyme is coupled to at least one polyethylene glycol (PEG).
8. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 7 , wherein the enzyme is coupled to the at least one PEG via one or more lysine residues.
9. A therapeutic formulation comprising the isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 2 , in a pharmaceutically acceptable carrier.
10. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 3 , wherein the enzyme is coupled to at least one polyethylene glycol (PEG).
11. The isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 10 , wherein the enzyme is coupled to the at least one PEG via one or more lysine residues.
12. A therapeutic formulation comprising the isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 3 , in a pharmaceutically acceptable carrier.
13. A composition comprising the isolated, modified human cystathionine-γ-lyase (CGL) enzyme of claim 1 , wherein the composition is at physiological pH.
14. The composition of claim 13 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme displays a k cat /K M for the hydrolysis of homocysteine greater than the wild-type human CGL enzyme.
15. The composition of claim 14 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme lacks a N-terminal methionine.
16. The composition of claim 13 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme displays a k cat /K M for the hydrolysis of homocysteine of about 11.9 s −1 mM −1 at pH 7.3 and 37° C.
17. The composition of claim 16 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme lacks a N-terminal methionine.
18. The composition of claim 13 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme displays a k cat /K M for the hydrolysis of homocysteine about 60-fold greater than wild-type human CGL.
19. The composition of claim 18 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme lacks a N-terminal methionine.
20. The composition of claim 13 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme displays a k cat /K M for the hydrolysis of homocystine of about 18.8 s −1 mM −1 at pH 7.3 and 37° C.
21. The composition of claim 20 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme lacks a N-terminal methionine.
22. The composition of claim 13 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme displays a k cat /K M for the hydrolysis of homocysteine of about 11.9 s −1 mM −1 at pH 7.3 and 37° C. and displays a k cat /K M for the hydrolysis of homocystine of about 18.8 s −1 mM −1 at pH 7.3 and 37° C.
23. The composition of claim 22 , wherein the isolated, modified human cystathionine-γ-lyase (CGL) enzyme lacks a N-terminal methionine.
24. A method of treating a subject having or at risk of developing homocystinuria or hyperhomocysteinemia comprising administering to the subject a therapeutically effective amount of the formulation of claim 6 .
25. The method of claim 24 , wherein the enzyme is coupled to at least one polyethylene glycol (PEG).
26. The method of claim 25 , wherein the enzyme is coupled to the at least one PEG via one or more lysine or cysteine residues.
27. The method of claim 24 , wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intramuscularly, intravesicularlly, intraumbilically, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.
28. The method of claim 24 , wherein the subject has previously been treated for homocystinuria or hyperhomocysteinemia and the enzyme is administered to prevent the recurrence of homocystinuria or hyperhomocysteinemia.
29. The method of claim 24 , further comprising administering at least a second homocystinuria or hyperhomocysteinemia therapy to the subject.
30. The method of claim 29 , wherein the second homocystinuria or hyperhomocysteinemia therapy is a high-dose vitamin B6 or betaine (N,N,N-trimethylglycine) therapy.
31. A method of treating a subject having or at risk of developing homocystinuria or hyperhomocysteinemia comprising administering to the subject a therapeutically effective amount of the formulation of claim 9 .
32. The method of claim 31 , wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intramuscularly, intravesicularlly, intraumbilically, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.
33. A method of treating a subject having or at risk of developing homocystinuria or hyperhomocysteinemia comprising administering to the subject a therapeutically effective amount of the formulation of claim 12 .
34. The method of claim 33 , wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intramuscularly, intravesicularlly, intraumbilically, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.