Recombinant human NaGlu protein and uses thereof
The present invention provides compositions comprising an isolated mixture of recombinant human NaGlu proteins in which a substantial amount of the NaGlu proteins in the mixture has increased levels of phosphorylated mannose that confer the proteins to be efficiently internalized into human cells. The present invention also provides methods of producing such mixture of NaGlu proteins, vectors used in transgenesis and expression, host cells harboring such vectors, and methods of isolating and purifying the mixture of NaGlu proteins. The invention further provides methods of treating NaGlu associated diseases.
1. A method of treating a subject suffering from NaGlu deficiency, the method comprising intravenously administering to the subject a therapeutically effective amount of a composition comprising an isolated recombinant human N-acetyl-alpha-D-glucosaminidase (rhNaGlu) whose amino acid sequence is set forth in 24-743 of SEQ ID NO:1, wherein said rhNaGlu comprises one or more glycan structures having sufficient amount of mannose-6-phosphate (M6P) that allows for internalization of said rhNaGlu into a mammalian cell having NaGlu deficiency via M6P receptor-mediated endocytosis, such that when internalized in vivo into a subject showing a deficiency in NaGlu, said rhNaGlu restores at least 50% of NaGlu activity observed in a cell of the same type in a normal subject, and wherein the rhNaGlu contains at least 1 mole of M6P per mole of the rhNaGlu protein, with the proviso that the rhNaGlu is not administered intrathecally.
2. The method of claim 1 , wherein said NaGlu deficiency is Sanfilippo syndrome B.
3. The method of claim 1 , wherein said subject is a human.
4. The method of claim 1 , wherein said recombinant human NaGlu protein is administered at a dosage of about 1 mg rhNaGlu per kg body weight to about 30 mg rhNaGlu per kg body weight.
5. The method of claim 1 , wherein said therapeutically effective amount is an amount effective to reduce heparan sulfate levels in the brain, the kidney, or the liver of the subject.
6. The method of claim 1 , wherein said therapeutically effective amount is an amount effective to increase NaGlu activity in the brain or the liver of the subject.
7. The method of claim 1 , further comprising administering a second therapeutic agent.
8. The method of claim 7 , wherein the second therapeutic agent is an immunosuppressant.
9. A method of delivering recombinant human NaGlu protein to the brain of a subject suffering from NaGlu deficiency, the method comprising intravenously administering recombinant human NaGlu protein (rhNaGLU) whose amino acid sequence is set forth in 24-743 of SEQ ID NO:1 to the subject, wherein the rhNaGlu comprises one or more glycan structures that contain mannose-6-phosphate (M6P) and contains at least 1 mole of M6P per mole of rhNaGlu protein, with the proviso that the rhNaGlu is not administered intrathecally.
10. The method of claim 1 , wherein the ratio of M6P to rhNaGlu protein is determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
11. The method of claim 9 , wherein the ratio of M6P to rhNaGlu protein is determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
12. A method of treating a subject suffering from Sanfilippo Syndrome B, comprising intravenously administering to the subject a therapeutically effective amount of a composition comprising recombinant human N-acetyl-alpha-D-glucosaminidase (rhNaGlu), wherein the amino acid sequence of said rhNaGlu is amino acids 24-743 of SEQ ID NO:1, the rhNaGlu comprises one or more glycan structures that contain mannose-6-phosphate (M6P) and contains about 1 to 6 moles of M6P per mole of rhNaGlu protein, with the proviso that the rhNaGlu is not administered intrathecally.
13. The method of claim 12 , wherein the therapeutically effective amount is about 1 mg rhNaGlu per kg body weight to about 5 mg rhNaGlu per kg body weight.
14. The method of claim 12 , wherein the therapeutically effective amount is about 5 mg rhNaGlu per kg body weight to about 30 mg rhNaGlu per kg body weight.
15. The method of claim 12 , wherein the therapeutically effective amount is about 5 mg rhNaGlu per kg body weight to about 10 mg rhNaGlu per kg body weight.
16. The method of claim 12 , wherein the therapeutically effective amount is about 5 mg rhNaGlu per kg body weight.
17. The method of claim 12 , wherein the therapeutically effective amount is about 10 mg rhNaGlu per kg body weight.
18. The method of claim 12 , wherein the ratio of M6P to rhNaGlu protein is determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).