Compositions for treating pathological calcification conditions, methods, and host cells using the same
The present invention includes compositions and methods for treating diseases or disorders associated with pathological calcification or pathological ossification. In certain embodiments, the diseases or disorders are selected from the group consisting of Generalized Arterial Calcification of Infancy (GACI), Idiopathic Infantile Arterial Calcification (IIAC), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, PXE, hereditary and non-hereditary forms of osteoarthritis, ankylosing spondylitis, hardening of the arteries occurring with aging, calciphylaxis resulting from end stage renal disease and progeria.
1. An isolated mammalian cell, comprising an ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) precursor polypeptide fusion;
wherein said ENPP1 precursor polypeptide fusion comprises:
a signal peptide, wherein the signal peptide is selected from the group consisting of an ectonucleotide pyrophosphatase/phosphodiesterase-5 (ENPP5) signal peptide and an ectonucleotide pyrophosphatase/phosphodiesterase-7 (ENPP7) signal peptide;
an ENPP1 polypeptide; and
a domain, wherein the domain is selected from the group consisting of albumin and immunoglobulin G (IgG) Fc; and
wherein said ENPP1 precursor polypeptide fusion is proteolytically processed upon secretion from the cell to yield a soluble enzymatically active ENPP1 polypeptide fusion comprising said ENPP1 polypeptide and said domain.
2. The isolated mammalian cell of claim 1 , wherein said ENPP1 precursor polypeptide fusion comprises a signal peptide selected from the group consisting of an ENPP5 signal peptide of human origin and an ENPP7 signal peptide of human origin.
3. The isolated mammalian cell of claim 1 , wherein said ENPP1 precursor polypeptide fusion comprises an ENPP1 polypeptide of human origin.
4. The isolated mammalian cell of claim 1 , wherein said soluble enzymatically active ENPP1 polypeptide fusion comprises residues 96-925 of human ENPP1 (SEQ ID NO: 1).
5. The isolated mammalian cell of claim 1 , wherein said ENPP1 precursor polypeptide fusion comprises a domain selected from the group consisting of serum albumin of human origin and IgG1 Fc of human origin.
6. The isolated mammalian cell of claim 1 , wherein said ENPP1 precursor polypeptide fusion further comprises polyaspartic acid residues.
7. A cell culture comprising the isolated mammalian cell of claim 1 .
8. The cell culture of claim 7 , wherein said cell culture comprises extracellular protein, and wherein said extracellular protein comprises said soluble enzymatically active ENPP1 polypeptide fusion comprising said ENPP1 polypeptide and said domain.
9. The cell culture of claim 8 , comprising 5 mg/liter of said ENPP1 polypeptide fused to said albumin.
10. The isolated mammalian cell of claim 5 , wherein said ENPP1 precursor polypeptide fusion comprises said ENPP1 polypeptide C-terminally fused to said human serum albumin, and wherein said ENPP1 precursor polypeptide fusion is proteolytically processed upon secretion from said cell to yield a soluble enzymatically active ENPP1 polypeptide fusion comprising said ENPP1 polypeptide C-terminally fused to said human serum albumin.
11. The isolated mammalian cell of claim 10 , wherein said human serum albumin comprises the sequence of SEQ ID NO: 27.
12. The isolated mammalian cell of claim 5 , wherein said ENPP1 precursor polypeptide fusion comprises said ENPP1 polypeptide C-terminally fused to said human IgG1 Fc, and wherein said ENPP1 precursor polypeptide fusion is proteolytically processed upon secretion from said cell to yield a soluble enzymatically active ENPP1 polypeptide fusion comprising said ENPP1 polypeptide C-terminally fused to said human IgG1 Fc.
13. The isolated mammalian cell of claim 12 , wherein said human IgG1 Fc comprises the sequence of SEQ ID NO: 26.
14. The isolated mammalian cell of claim 2 , wherein said ENPP7 signal peptide comprises residues 1-22 of ENPP7 (SEQ ID NO: 17).
15. The isolated mammalian cell of claim 2 , wherein said ENPP5 signal peptide comprises residues 1-24 of ENPP5 (SEQ ID NO: 24).
16. The isolated mammalian cell of claim 3 , wherein said ENPP1 precursor polypeptide fusion lacks residues 77-98 of human ENPP1 (SEQ ID NO: 1).
17. The isolated mammalian cell of claim 3 , wherein said ENPP1 precursor polypeptide fusion comprises residues 1-76 of human ENPP1 (SEQ ID NO: 1) and residues 96-925 of human ENPP1 (SEQ ID NO: 1).