DNASE I POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME, METHODS OF PRODUCING DNASE I AND USES THEREOF IN THERAPY
Plant-expressed human recombinant DNase proteins, nucleic acid constructs for expression of the human recombinant DNase I in plant cells, cells expressing the nucleic acid construct and therapeutic uses thereof are disclosed. Particularly, compositions and methods for treating pulmonary and/or respiratory conditions by inhalation of the plant-expressed human recombinant DNase I are provided.
1 . An isolated polynucleotide comprising a nucleic acid sequence encoding a human DNase I protein, wherein said human DNase I protein is translationally fused at the N-terminus to a plant endoplasmic reticulum targeting signal peptide.
2 - 11 . (canceled)
12 . A nucleic acid construct comprising the isolated polynucleotide of claim 1 .
13 . A cell comprising the nucleic acid construct of claim 12 .
14 - 18 . (canceled)
19 . A method of producing a recombinant human DNase I protein, comprising:
providing a cell according to claim 13 ; and
growing said cell so as to produce said recombinant human DNase I protein.
20 . The method of claim 19 , further comprising isolating said recombinant human DNase I protein from said cell.
21 . (canceled)
22 . The method of claim 19 , further comprising isolating said recombinant human DNase I protein from a plant cell culture medium.
23 . (canceled)
24 . An isolated human DNase I protein having an N-terminal Glycine residue.
25 . The human DNase I protein of claim 24 , having an amino acid sequence as set forth in SEQ ID NO: 5.
26 . The human DNase I protein of claim 25 , having at least one core xylose and at least one core α-(1,3) fucose.
27 . The human DNase protein of claim 24 , having reduced susceptibility to actin inhibition of endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I.
28 . The human DNase protein of claim 24 , having lower Michaelis constant (K m ) and greater reaction velocity (Vmax) for endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I.
29 . A human DNase I protein wherein said DNase I protein is translationally fused at the N-terminus to a plant endoplasmic reticulum targeting signal peptide.
30 . The human DNase I protein of claim 29 , wherein said plant endoplasmic reticulum targeting signal peptide is an Arabidposis ABPI signal.
31 . The human DNase I protein of claim 29 , having an amino acid sequence as set forth in SEQ ID NO: 1.
32 . A pharmaceutical composition comprising, as an active ingredient, the human DNase I protein of claim 24 and a pharmaceutically acceptable carrier.
33 . The pharmaceutical composition of claim 32 , further comprising, as an active ingredient, a magnesium salt.
34 . The pharmaceutical composition of claim 32 , further comprising, as an active ingredient, an agent for inhibiting formation of G actin and/or enhancing formation of F actin.
35 - 36 . (canceled)
37 . The pharmaceutical composition of claim 32 , wherein said human DNase I protein is at least 90-95% pure human DNase I protein.
38 . The pharmaceutical composition of claim 32 , further comprising plant beta-acetylhexosaminidase enzyme protein.
39 . (canceled)
40 . A method of reducing viscous modulus or elastic modulus of sputum in a subject in need thereof, the method comprising administering to the subject the human DNase I protein of claim 24 , thereby reducing the viscous modulus or elastic modulus of sputum in the subject.
41 . (canceled)
42 . A method of treating a DNase-I related disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 32 , thereby treating the DNase-I related disease or condition in the subject.
43 . The method of claim 42 , wherein said disease or condition is associated with excess extra-cellular DNA in a fluid, secretion or tissue of said subject, and wherein said pharmaceutical compositions causes nucleolysis of said extra-cellular DNA.
44 . The method of claim 42 , wherein said human DNase I protein is formulated for administration via a nebulizer.
45 . The method of claim 42 , wherein said disease or condition is a pulmonary disease or condition.
46 - 50 . (canceled)