(19)
(11)EP 2 809 780 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
02.05.2018 Bulletin 2018/18

(21)Application number: 13711558.0

(22)Date of filing:  31.01.2013
(51)International Patent Classification (IPC): 
A61K 38/46(2006.01)
A61K 45/06(2006.01)
A61K 33/06(2006.01)
C12N 15/82(2006.01)
A61K 9/14(2006.01)
A61K 38/47(2006.01)
A61K 9/00(2006.01)
A61K 33/14(2006.01)
C12N 9/22(2006.01)
(86)International application number:
PCT/IL2013/050097
(87)International publication number:
WO 2013/114374 (08.08.2013 Gazette  2013/32)

(54)

DNASE I POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME, METHODS OF PRODUCING DNASE I AND USES THEREOF IN THERAPY

INHALIERBARE FLÜSSIGE FORMULIERUNGEN DER DNASE I

COMPOSITION LIQUIDE INHALABLE DE DNASE I


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 01.02.2012 US 201261593350 P
01.02.2012 US 201261593788 P
01.02.2012 US 201261593676 P

(43)Date of publication of application:
10.12.2014 Bulletin 2014/50

(73)Proprietor: Protalix Ltd.
2010000 Carmiel (IL)

(72)Inventors:
  • SHAALTIEL, Yoseph
    3657600 Timrat (IL)
  • HANANIA, Uri
    2199034 Carmiel (IL)
  • KIZHNER, Tali
    2017000 Doar-Na Misgav (IL)
  • MATIUHIN, Yulia
    3710502 Pardes Chana - Karkur (IL)
  • FUX, Liat
    2623183 Kiryat-Motzkin (IL)
  • SHULMAN, Avidor
    2017500 Doar-Na Misgav (IL)

(74)Representative: Vogel, Andreas et al
Bals & Vogel Universitätsstrasse 142
44799 Bochum
44799 Bochum (DE)


(56)References cited: : 
EP-A1- 2 468 870
WO-A1-93/25670
WO-A2-2011/053982
US-A1- 2006 228 347
WO-A1-90/07572
WO-A1-2004/003207
CN-A- 1 458 161
US-A1- 2011 237 782
  
  • SOJIKUL PUNCHAPAT ET AL: "A plant signal peptide-hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 100, no. 5, 4 March 2003 (2003-03-04) , pages 2209-2214, XP002473534, ISSN: 0027-8424, DOI: 10.1073/PNAS.0438037100
  • D. KL MBT: "Auxin-binding Protein 1 Does Not Bind Auxin within the Endoplasmic Reticulum Despite This Being the Predominant Subcellular Location for This Hormone Receptor", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 45, 10 November 1995 (1995-11-10), pages 26962-26969, XP055065756, ISSN: 0021-9258, DOI: 10.1074/jbc.270.45.26962
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

FIELD AND BACKGROUND OF THE INVENTION



[0001] The present invention, in some embodiments thereof, relates to a plant nucleic acid expression construct and, more particularly, but not exclusively, to a nucleic acid construct for expression of DNase I in plant cells, cells expressing the nucleic acid construct, plant-expressed DNase I and therapeutic uses thereof.

[0002] Human DNase I is a member of the mammalian DNase I family (EC 3.1.21.1). DNase I belongs to the class of Mg2+ and Ca2+ dependent endonucleases, whose hydrolytic activity depends on the presence of bivalent metals. Magnesium ion is involved in electrophilic catalysis of the phosphodiester bond cleavage, whereas Ca2+ maintains optimal enzyme conformation. DNase I cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. It acts on single-stranded DNA, double-stranded DNA, and chromatin.

[0003] The principal therapeutic use of human DNase has been to reduce the viscoelasticity of pulmonary secretions (including mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. Mucus also contributes to the morbidity of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold. DNase I is effective in reducing the viscoelasticity of pulmonary secretions by hydrolyzing high-molecular-weight DNA present in pulmonary secretions. DNase has also been proposed for non-pulmonary disorders, for example, treatment of male infertility and uterine disorders (see US 2007/0259367), inhibition of metastatic growth (see US 7,612,032) and applied topically for viral conditions.

[0004] Following initial dissatisfaction with naturally occurring DNase I in the clinical setting (mostly due to co-purified contaminating proteases and immunogenicity of bovine DNase), DNA encoding human DNase I was isolated and sequenced, and expressed in recombinant host cells, thereby enabling the production of human DNase in commercially useful quantities. Recombinant human DNase (rhDNase) (e.g. dornase alfa; Pulmozyme®, Genentech., CA), expressed in Chinese hamster ovary (CHO) cells, has been found to be clinically effective for CF.

[0005] Recombinant human DNase I for therapeutic use (dornase alfa; Pulmozyme®) is currently produced in mammalian cells (CHO cells). Expression of recombinant DNase I has also been described in human embryonic kidney 293 cells (Shak et al, PNAS 1990;87:9188-92), and in bacteria. Expression of fusion proteins including human recombinant DNase I has also been suggested in the protozooan Tetrahymena expression system (Weide et al, BMC Biotech 2006;6:19), in plant or algal plastids (US Patent Application No: 2011/151515 to Heifetz et al), and in a variety of other host cells (see, for example, US Patent Application No: 2007/0037257 to Chisholm et al; US Patent Application 2005/0019925 to Krummer et al and US Patent Application No. 2004/0077013 to Ashkenazi et al). Recombinant human DNase (rhDNase) (e.g. dornase alfa; Pulmozyme®, Genentech., CA), expressed in Chinese hamster ovary (CHO) cells was approved for clinical use by the FDA in 1994, and additional human clinical trials with recombinant human DNase I are currently in progress for CF (see NCT01155752; NCT0017998; NCT00117208 and NCT00204685) and other chronic, respiratory diseases such as atelectasis (see NCT01095276 and NCT00671323) and Sjogren's Syndrome. As of 2011, thousands of patients among the approximately 30,000 patient population in the US, and around the world, have been treated with recombinant human DNase I, and in general, safety and efficacy of both have been demonstrated.

[0006] Human enzymes can be produced in transgenic plants in order to address problems of safety, viral infections, immune reactions, production yield and cost. US Patent No. 6,391,638 and PCT WO2008/135991 teach bioreactor devices for commercial-scale production of recombinant polypeptides from plant cell culture. US Patent No. 7,951,557, US Patent Application Nos. 10/554,387 and 11/790,991 teach construction and expression of nucleic acid vectors for recombinant expression of human enzymes in plant cells. PCT WO2007/010533 teaches the expression of recombinant human polypeptides, including DNase I, in plant cells, for enteral administration.

SUMMARY OF THE INVENTION



[0007] According to an aspect of some embodiments of the present invention there is provided 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.

[0008] According to some embodiments of the present invention a codon usage of the nucleic acid sequence is optimized for Nicotinia tabaccum.

[0009] According to some embodiments of the present invention the human DNase I comprises an N-terminal Glycine residue.

[0010] According to some embodiments of the present invention the human DNase I protein comprises the amino acid sequence as set forth in SEQ ID NO: 6.

[0011] According to some embodiments of the present invention the nucleic acid sequence encoding the human DNase I protein comprises SEQ ID NO: 9.

[0012] According to some embodiments of the present invention the human DNase I protein comprises the amino acid sequence as set forth in SEQ ID NO: 5.

[0013] According to some embodiments of the present invention the nucleic acid sequence encoding the human DNase I protein comprises SEQ ID NO: 277.

[0014] According to some embodiments of the present invention the isolated polynucleotide comprises a nucleic acid sequence encoding the endoplasmic reticulum targeting signal peptide as set forth in SEQ ID NO: 10.

[0015] According to some embodiments of the present invention the nucleic acid sequence has a sequence as set forth in SEQ ID NO: 12.

[0016] According to some embodiments of the present invention the human DNase I protein has an amino acid sequence as set forth in SEQ ID NO: 1 or as set forth in SEQ ID NO: 5.

[0017] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the isolated polynucleotide of the invention, the 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.

[0018] According to an aspect of some embodiments of the present invention there is provided a cell comprising the nucleic acid construct comprising the isolated polynucleotide of the invention.

[0019] According to some embodiments of the present invention the cell is a plant cell.

[0020] According to some embodiments of the present invention the plant cell is a tobacco cell.

[0021] According to some embodiments of the present invention the tobacco cell is a BY-2 cell.

[0022] According to some embodiments of the present invention the tobacco cell is a BY2 tobacco cell in suspension.

[0023] According to some embodiments of the present invention the nucleic acid construct is stably integrated into the genome of said cell.

[0024] According to an aspect of some embodiments of the present invention there is provided a method of producing a recombinant human DNase I protein, comprising:

providing a cell according to invention; and

growing the cell so as to produce said recombinant human DNase I protein.



[0025] According to some embodiments of the present invention the method further comprises isolating the recombinant human DNase I protein from the cell.

[0026] According to some embodiments of the present invention the cell is an isolated cell cultured in a plant cell culture medium.

[0027] According to some embodiments of the present invention the method further comprises isolating the recombinant human DNase I protein from the plant cell culture medium.

[0028] According to some embodiments of the present invention the culturing is affected in a disposable bioreactor.

[0029] According to an aspect of some embodiments of the present invention there is provided an isolated human DNase I protein having an N-terminal Glycine residue.

[0030] According to some embodiments of the present invention the isolated protein has an amino acid sequence as set forth in SEQ ID NO: 5.

[0031] According to some embodiments of the present invention the human DNase I protein, has at least one core xylose and at least one core α-(1,3) fucose.

[0032] According to some embodiments of the present invention the human DNase protein has reduced susceptibility to actin inhibition of endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I.

[0033] According to some embodiments of the present invention the human DNase protein has a lower Michaelis constant (Km) and greater reaction velocity (Vmax) for endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I.

[0034] According to an aspect of some embodiments of the present invention there is provided a human DNase I protein wherein the DNase I protein is translationally fused at the N-terminus to a plant endoplasmic reticulum targeting signal peptide.

[0035] According to some embodiments of the present invention the plant endoplasmic reticulum targeting signal peptide is an Arabidposis ABPI signal.

[0036] According to some embodiments of the present invention the human DNase I protein has an amino acid sequence as set forth in SEQ ID NO: 1.

[0037] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the human DNase I protein of the invention, the DNase I protein being translationally fused at the N-terminus to a plant endoplasmic reticulum targeting signal peptide, and a pharmaceutically acceptable carrier.

[0038] According to some embodiments of the present invention the pharmaceutical composition further comprises, as an active ingredient, a magnesium salt.

[0039] According to some embodiments of the present invention the pharmaceutical composition further comprises, as an active ingredient, an agent for inhibiting formation of G actin and/or enhancing formation of F actin.

[0040] According to some embodiments of the present invention the pharmaceutical composition is formulated for delivery via nebulizer.

[0041] According to some embodiments of the present invention the human DNase I protein is at least 90-95% pure human DNase I protein.

[0042] According to some embodiments of the present invention the pharmaceutical composition further comprising plant beta-acetylhexosaminidase enzyme protein. In some embodiments the plant beta-acetylhexosaminidase enzyme protein is inactivated beta-acetylhexosaminidase enzyme protein. In some embodiments the beta-acetylhexosaminidase enzyme protein is heat inactivated beta-acetylhexosaminidase enzyme protein.

[0043] According to an aspect of some embodiments of the present invention there is provided a method of reducing the viscous modulus of sputum, the method comprises exposing the sputum to an effective concentration of the human DNase I protein of the invention.

[0044] According to an aspect of some embodiments of the present invention there is provided a method of reducing the elastic modulus of sputum, the method comprising exposing the sputum to an effective concentration of the human DNase I protein of the invention.

[0045] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the invention.

[0046] According to some embodiments of the present invention disease or condition is associated with excess extra-cellular DNA in a fluid, secretion or tissue of said subject, and said administering said pharmaceutical compositions results in nucleolysis of said extra-cellular DNA.

[0047] According to some embodiments of the present invention administering the pharmaceutical composition is effected via nebulizer.

[0048] According to some embodiments of the present invention the subject is suffering from a pulmonary disease or condition.

[0049] According to some embodiments of the present invention the pulmonary disease or condition is selected from the group consisting of acute or chronic bronchopulmonary disease, atelectasis due to tracheal or bronchial impaction, and complications of tracheostomy.

[0050] According to some embodiments of the present invention the acute or chronic bronchopulmonary disease is selected from the group consisting of infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, TB or fungal infections.

[0051] According to an aspect of some embodiments of the present invention there is provided a method of treating Cystic Fibrosis in subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the invention.

[0052] According to some embodiments of the present invention the subject is suffering from a disease or condition selected from the group consisting of male infertility, metastatic cancer, a viral, bacterial, fungal or protozoan infection sepsis, atherosclerosis, diabetes, delayed type hypersensitivity and a uterine disorder.

[0053] According to some embodiments of the present invention the pharmaceutical composition is formulated for administration via a method selected from the group consisting of inhalation, direct local administration, parenteral administration, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration and mucosal administration.

[0054] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS



[0055] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0056] In the drawings:

FIG. 1 is an amino acid sequence alignment of the plant recombinant human DNase I (prhDNase I) encoded by the nucleic acid of the invention [SEQ ID NO: 1, sequence (b)], and native human DNase I protein [(GenBank: NM_005223, sequence (a)](SEQ ID NO: 2), including the native signal leader peptide SEQ ID NO: 3). The Arabidopsis ABPI endoplasmic reticulum targeting signal peptide (SEQ ID NO: 4) is highlighted;

FIG. 2 is a graph showing the results of targeting expression of recombinant human DNase I to different plant organelles. rhDNase I was expressed in whole tobacco plants and targeted for secretion (to the apoplast) via an N-terminal ER targeting signal peptide (APO), targeted to the vacuole, via an N-terminal ER targeting signal and a C-terminal vacuolar targeting signal peptide (VAC) and targeted to the endoplasmic reticulum via an N-terminal ER targeting signal peptide and a C-terminal ER retention signal peptide (ER). Activity of rhDNase I in the cells of the tobacco plant was monitored by immunoreactivity (ELISA) and according to catalytic activity (DNA-methyl green assay), adjusted for amount of biomass in sample and expressed as phrDNase I relative to expression in apoplast-targeted cells (100%). Note the significantly greater yield when the rhDNase I is targeted to the apoplast;

FIG. 3 shows an SDS-PAGE gel illustrating the size and purity of plant-expressed rh DNase I. 1.25 µg, 2.5 µg, 5 µg and 10 µg of commercial human DNase (Pulmozyme®) (lanes 1-4, respectively) or purified prh DNase I (lanes 6-9, respectively) were separated on 15% Tris-Glycine SDS-PAGE, stained with Coomassie blue, and analyzed in comparison to molecular weight standards (lanes 5 and 10). Note the single, predominant band of the plant-expressed enzyme migrating slightly but discernibly faster than that of commercial human DNase (Pulmozyme®), and the absence of detectable protein impurities;

FIG. 4 shows an SDS-PAGE gel illustrating the size, purity and antigenic identity of plant-expressed rh DNase I. 10 ng, 50 ng, 100 ng and 200 ng of commercial human DNase (Pulmozyme®) (lanes 1-4, respectively) or purified prh DNase I (lanes 6-9, respectively) were separated on 15% Tris-Glycine SDS-PAGE, transferred to nitrocellulose membrane, probed with whole antiserum primary antibody obtained from rabbits immunized against Pulmozyme®, and visualized with secondary goat anti- rabbit IgG HRP-conjugated antibody. Note the single, predominant band of the immunoreactive, plant-expressed enzyme migrating and reacting similarly to that of commercial human DNase (Pulmozyme®), and the absence of detectable protein impurities;

FIGs. 5A-5D shows an IEF gel and capillary electropherogram of human DNase (Pulmozyme®) and purified, plant expressed rh DNase I. Figure 5A shows the resolution of 6µg of commercial human DNase (Pulmozyme®) (lane 1) or 6µg of purified, plant expressed rh DNase I (lanes 2-3) on an isoelectric focusing gel. Figure 5B shows the resolution of 8µg of commercial human DNase (Pulmozyme®) (lane 3) or 8µg of purified, plant expressed rh DNase I from a different line of BY2 cells expressing the prhDNase I (lane 2) on an isoelectric focusing gel. Note that Pulmozyme® resolves into multiple bands with isoelectric points (pI) between pI 3.5 and pI 4.5 in a pH gradient of pH 3 to pH 7, while in both of the purified, plant expressed rh DNase I preparations the DNase I resolved at an isoelectric point (pI) between pI 4.2 and pI 4.5, in two major bands and one minor band. Electrophoresis conditions included 100m V-1 hour, 200mV-1 hour and 500mV-1.5 hours. Lanes 4 (Figure 5A) and lane 1 (Figure 5B) contain protein standards for comparison. Figure 5D is an electropherogram of image capillary isoelectric focusing (scale = pI 3.3-6.1) analysis of purified, plant expressed rh DNase I, showing the resolution of the prh DNase I as observed in the IEF gel [one major peak (3) at pI 4.41 and two minor peaks (4 and5) at pI 4.27 and 4.21). 1 and 2 are pI markers 5.85 and 3.59. Figure 5C is an electropherogram of the blank scale with pI markers 1 and 2;

FIGs. 6A-6B show the molecular mass analysis of purified, plant expressed rh DNase I by mass spectrometry using 2.5 micrograms of the purified prh DNase I using a matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometer, with sinapinic acid as a matrix. Figure 6A showing the entire spectrum from 20000 to 180000 m/z and Figure 6B showing an enlarged segment of the prh-DNaseI peak at about 32000 m/z;

FIG. 7 is a figure representing a putative amino acid sequence of the purified, plant expressed rh DNase I (SEQ ID NO: 5), derived from the sequences of overlapping peptides produced by partial proteolytic digestion (see SEQ ID NOs: 17-276) and separated and analyzed on RP-HPLC and mass spectrometry. Unconfirmed amino acids are marked, glycosylation sites are underlined and the N-terminal Glycine residue is marked;

FIGs. 8A-8C represent analysis of the glycan structure of the purified, plant expressed rh DNase I, as determined by exoglucosidase digestion and NP-HPLC analysis of the resulting de-glysosylated polypeptides. FIG. 8A is an NP-HPLC profile of total glycans released following PNGase A digestion of the glycosylated prhDNase I, derived from three separate exemplary batches of prhDNase I. The numbers above the discrete peaks correspond to specific glycan structures illustrated in FIG. 8B. FIG 8B is a chart showing individual glycan structures released from prhDNase I by PNGase A digestion, and their relative abundance (in percentages) from total released glycans, for each of the three exemplary batches tested. Note the predominance of two main glycan peaks representing a high percentage of glycan structures (over 80%) containing mannose 3- β-(1,2) xylose- α-(1,3) fucose [Fc(3)M3X] and/or mannose 4- α-(1,2) xylose. FIG. 8C shows an NP-HPLC profile of total glycans released from prhDNase I using PNGase A endoglycosidase (top) compared to the profile of N-linked glycans released from Pulmozyme® using PNGase F endoglycosidase (specific to high mannose glycans) showing a multitude of glycan variations. The major peaks are annotated with corresponding glycan structures. Note the wide variation in glycosylation pattern found in Pulmozyme®, due to the abundance of bi- and tri-antennary glycans, and sialic acid residues.

FIGs. 9A-9D are chromatograms of RP-HPLC of purified, plant expressed rh DNase I, analyzed at 214 nm (FIG. 9A, 9B) and 280 nm (FIG. 9C, 9D), indicating a preparation of the prh DNase I having less than 7% impurities (93.77% pure measured at 214nm, and 93.62% pure measured at 280 nm). Insets (FIGs. 9B and 9D) are expanded views of the prh DNase I peak at 214 nm and 280 nm, respectively;

FIGs. 10A-10B are graphs representing substrate inhibition kinetic plots of purified prh DNase I (open circles O) and commercial human DNase (Pulmozyme®) (closed circles •), using the DNaseAlert ™ fluorometric substrate detected at 535 nm and 565 nm. FIG. 10A is a plot of the initial velocity as a function of substrate concentration. Note the superior kinetics (greater Vmax, lower KM, e.g. higher specific activity) of the purified prh DNase I. FIG. 10B is a double reciprocal plot of initial velocity as a function of substrate concentration. KM and Vmax values were calculated using the following linear regression equations (plotted at substrate concentration of 2-15 µM) and R2 values: prh DNase I : y = 41.377x + 1.8693, R2 = 0.9894; Pulmozyme®: y = 120.17x + 4.8316, R2 = 0.9921, indicating substrate-inhibition-like kinetics for both prh DNase I and Pulmozyme®;

FIG. 11 is an activity plot of purified, plant expressed rh DNase I (open circles O) and commercial human DNase (Pulmozyme®) (closed circles •), illustrating resistance to actin inhibition of DNase I activity. ΔOD (620 nm) is plotted as a function of G-actin concentration, using the Methyl Green substrate, to yield IC50 values. Note the reduced susceptibility to G-actin inhibition of the purified prh DNase I, compared to that of commercial human DNase (Pulmozyme®);

FIGs. 12A-D are histograms illustrating the effect of increasing concentrations of DNase I on the elastic modulus of sputum from Cystic Fibrosis patients, determined using time-sweep measurements with a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany). FIG. 12A represents a comparison of the effect of purified, plant expressed rh DNase I (dark bars) and commercial human DNase (Pulmozyme®) (hatched bars) on the elastic modulus of CF patient sputa, measured at 2, 10 and 20 µg DNase I /gr sputum. DNA content of the sputum was 4.66 µg DNA/ gr sputum. Each value represents at least 2 determinations measured from each sputum sample. FIGs. 12B-12D show the effect of prhDNase I on the elastic modulus of sputa collected from individual patients. Note the pronounced, superior and consistent, dose-dependent reduction of sputum elastic modulus following incubation with prh DNase I, as compared to the Pulmozyme®;

FIGs. 13A-D are histograms illustrating the effect of increasing concentrations of DNase I on the viscous modulus of sputum from Cystic Fibrosis patients, measured using time-sweep technique with a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany). FIG. 13A represents a comparison of the effect of purified, plant expressed rh DNase I (dark bars) and commercial human DNase (Pulmozyme®) (hatched bars) on the viscous modulus of CF patient sputa, measured at 2, 10 and 20 µg DNase I /gr sputum. DNA content of the sputum was 4.66 µg DNA/ gr sputum. Each value represents at least 2 determinations measured from each sputum sample. FIGs. 13B-13D shows the effect of prhDNase I on the viscous modulus of sputa collected from individual patients. Note the pronounced, superior and consistent, dose-dependent reduction of sputum viscous modulus following incubation with prh DNase I, as compared to the Pulmozyme®;

FIGs. 14A and 14B are histograms representing the effect of prhDNase I on the rheological properties of sputum from Cystic Fibrosis patients, measured using time sweep technique, expressed as percent change rheological properties (elastic modulus, FIG. 14A and viscous modulus, FIG. 14B) compared to an untreated sample. FIG. 14A shows the percent change of elastic modulus of the sputa of 4 individual patients (black bars= untreated control samples; hatched bars= 2 µg prhDNase I; gray bars= 20 µg prhDNase I). FIG. 14B shows the percent change of viscous modulus of the sputa of 4 individual patients (black bars= untreated control samples; hatched bars= 2 µg prhDNase I; gray bars= 20 µg prhDNase I). Note the consistent pronounced reduction in rheological properties following incubation of the sputa with prh DNase I;

FIG. 15 is a histogram illustrating the synergic effect of purified, plant expressed rh DNase I on the reduction of CF sputum elastic modulus by Magnesium (MgCl2) (0, 25, 50 and 100 mM), measured using time sweep technique, with rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany). Elastic modulus of the sputum was determined at indicated concentrations of Mg2+ (mM MgCl2) in the presence (cross-hatched bars) or absence (dark bars, buffer only) of prh DNase I (2 µg prh DNase I /gr sputum). Each value represents at least 2 determinations. Note the synergic reduction of sputum elastic modulus following incubation with the prh DNase I and Magnesium, as compared to Magnesium alone;

FIG. 16 is a histogram illustrating the effect of Magnesium salts on DNase I reduction of CF sputum viscous modulus. Figure 16 shows the synergic effect of purified, plant expressed rh DNase I on the reduction of CF sputum viscous modulus by Magnesium (MgCl2) (0, 25, 50 and 100 mM), measured with a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany). Viscous modulus of the sputum was determined at the indicated concentrations of Mg2+ (mM MgCl2) in the presence (cross hatched bars) or absence (grey bars, buffer only) of prh DNase I (2 µg DNase I /gr sputum). Each value represents at least 2 determinations. Note the synergic reduction of sputum viscous modulus following incubation with the prh DNase I and Magnesium, as compared to Magnesium alone;

FIG. 17 is a graph illustrating the effect of different Magnesium salts on the DNase catalytic activity of purified, plant expressed rh DNase I. Catalytic activity of rh DNase I was measured in the presence of increasing concentrations (0.5-100 mM) of Magnesium chloride (MgCl2, closed circles •) or Magnesium sulphate (MgSO4, open diamonds ◊), using the methyl green substrate and expressed as the change in absorbance at 630 nm (Δ OD 630 nm) after 3 hours incubation with the enzyme. Note that neither magnesium salt inhibited rh DNase I activity up to 50 mM, and that rh DNase I activity was only slightly impaired by MgSO4 at 100 mM.

FIGs. 18A-18D are histograms illustrating the effect of increasing concentrations of DNase I on the rheological properties of sputum from Cystic Fibrosis patients, determined using the stress sweep technique with a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany), expressed as the stress (Pa) at cross-over of elastic and viscous modules (phase angle = 45°). FIGs. 18A-18D represents a comparison of the effect of purified, plant expressed rh DNase I (dark bars) and commercial human DNase (Pulmozyme®) (hatched bars) on the rheological properties of four individual CF patient sputa, measured at 0.2, 2 and 20 µg DNase I /gr sputum. DNA content of the sputum was 3.09 mg DNA/ gr sputum, 3.15 mg DNA/ gr sputum, 8.36 mg DNA/ gr sputum and 3.89 mg DNA/ gr sputum (FIGs. 18A-18D, respective). Each value represents at least 2 determinations measured from each sputum sample. Note the pronounced and consistent, dose-dependent reduction in cross-over stress values following incubation with prh DNase I;

FIGs. 19A to 19D are histograms representing the effect of prhDNase I and Pulmozyme® on the rheological properties of sputum from four different Cystic Fibrosis patients (19A-19D), measured using stress sweep technique, expressed as the stress (Pa) at cross-over of elastic and viscous modules (phase angle = 45°). DNA content of the sputum was 2.16 mg DNA/ gr sputum, 2.63 mg DNA/ gr sputum, 3.45 mg DNA/ gr sputum and 4.17 mg DNA/ gr sputum (FIGs. 19A-19D, respective) Black bars= prhDNase I; hatched bars= Pulmozyme®. DNase I concentration (0, 2 or 20 µg DNase I/ gr sputum). Note the consistent pronounced reduction in rheological properties following incubation of the sputa with prh DNase I, significantly outperforming Pulmozyme® in 3 out of the four patients;

FIGs. 20A-20C are histograms illustrating the effect of Magnesium salts and DNase I on the rheological properties of CF sputum samples from three different Cystic Fibrosis patients, measured using the stress sweep technique with a rheomoter (HAAKE RheoStress I, Thermo Fisher Scientific GmBH, Germany), expressed as the stress (Pa) at cross-over of elastic and viscous modules (phase angle = 45°). FIG. 20A-patient A (1.84 mgDNA/g sputum), FIG. 20B- patient B (3.46 mgDNA/g sputum) and FIG. 20C (2.39 mg DNA/g sputum). Dark columns are measurements without Magnesium, grey columns are represent measurement with 100 mM MgSO4, measured at 0 (control) 2 and 20 µg DNase I /gr sputum. Each value represents at least 2 determinations measured from each sputum sample. Note the pronounced and synergistic disruption of sputum elastic structure following incubation with prh DNase I and MgSO4.


DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION



[0057] The present invention, in some embodiments thereof, relates to a an isolated polynucleotide encoding a human DNase I polypeptide, a plant expression vector for recombinant expression of the human DNase I polypeptide in plant cells, methods for the expression thereof in plant cells and the human DNase I polypeptide produced thereby. In particular, the present invention relates to therapeutic use of the plant-expressed human DNase I for pulmonary and other disorders, such as Cystic Fibrosis.

[0058] It is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0059] Clinical experience, beginning with bovine pancreatic DNase I, has indicated that the available recombinant enzyme compositions may differ with regard to patient tolerance, immunogenicity, dosage regimen and clinical efficacy. Another drawback associated with the existing recombinant enzymes is their expense, which can place a heavy economic burden on health care systems. Genentech's revenues from Pulmozyme® have topped 300 million dollars, annually, for more than three years since 2008. The high cost of these recombinant enzymes, when produced in mammalian cell cultures or bioreactors results from a complex purification and modification protocol, and the relatively large amounts of the therapeutic required for existing treatments.

[0060] Further, yields and clinical effectiveness of some recombinant proteins, among them therapeutically important ones such as enzymes, have often been disappointing in the past. Still further, judging by the absence of new clinical trials, few or no candidates for the "next generation" of clinically suitable human recombinant DNase I have come to the fore. Effective human DNase I production in a plant cell-based expression system, for increased efficiency of expression and production of recombinant human DNase I having improved catalytical activity and pharmacokinetics under clinical conditions could significantly reduce the cost of DNase I so that this life saving therapy can be provided more affordably to all who require it.

[0061] Further, the present inventors have also attempted to address the phenomenon of actin inhibition of endonuclease catalytic activity, which threatens to reduce the efficacy of DNase I administration for respiratory diseases and other conditions associated with mucus accumulation. Along with DNA released from cells following neutrophil activity in bronchopulmonary disease, actin accumulates, further contributing to the viscidity of the resultant mucus. Inhibition of DNase catalytic activity by actin has been documented, and is critical to the effectiveness of DNase inhalation therapy. Studies of actin-DNase I interaction have revealed that the G-form of actin is highly inhibitory of DNase I activity, while the F-actin form is not.

[0062] The present inventors have screened for a plant expression vector for recombinant expression of human DNase I in plant cells comprising a polynucleotide encoding a human DNase I polypeptide, transformed tobacco plants with the vector, and have isolated catalytically active human DNase I from the plants. The biochemical properties of the plant-expressed recombinant human DNase I compare favorably with those of the commercially available clinical standard Pulmozyme ® (Dornase alpha), and results with CF sputum suggest an advantageous effect of the plant-expressed recombinant human DNase I on the reduction of rheological parameters of sputum, as well as reduced susceptibility to actin inhibition of the DNase endonuclease activity.

[0063] Structural differences uncovered between the plant-expressed recombinant human DNase I and mammalian-cell expressed recombinant human DNase I (e.g. Pulmozyme ®) (mobility on PAGE, unity of immunoreactive DNase I species as detected on Western blots, see hereinbelow) suggest that both modifications of amino acid sequence and post translational modification resulting from processing of the expressed polypeptide by the plant cell may be responsible for some of the distinguishing functional characteristics of the plant-expressed recombinant enzyme.

[0064] Thus, according to one aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a human DNase I protein, wherein the human DNase I protein is contiguously linked at the N-terminal to a plant endoplasmic reticulum targeting signal peptide.

[0065] As used herein the term "human DNase I protein" refers to a human DNase I (deoxyribonuclease I; EC 3.1.21.1; DNase I) polypeptide. Human DNase I is classified as an endonuclease, which cleaves DNA to produce 5' phosphodi- and 5' phosphooligonucleotide end products, with a preference for double stranded DNA substrates and alkaline pH optimum. Other members of the DNase I family of endonucleases are DNase X, DNase lambda, DNASIL2 and tear lipocalin in humans. DNase I is also known, inter alia, as alkaline DNase, bovine pancreatic (bp) DNase, DNase A, DNA phosphatase and DNA endonuclease, for example, in Bos taurus. According to some embodiments of the invention, the human DNase I protein is the mature human DNase I protein, having the amino acid sequence as set forth in SEQ ID NO: 6. It will be appreciated that the human DNase I protein can be a modified human DNase I protein, having an amino acid sequence different than that of SEQ ID NO: 6 while maintaining characteristic structure and/or function of DNase I. One non-limiting example of a modified human DNase I protein encoded by the expression vector of the invention is SEQ ID NO: 5.

[0066] According to yet other embodiments of the invention, the plant-expressed human DNase protein is a variant human DNase protein. Variant human DNase proteins, having altered catalytic and/or other biochemical and structural properties, such as altered actin affinity, cofactor requirements, pH optimum, increased shelf life in storage and the like, enhanced recombinant expression or fusion proteins have been disclosed (see, for example, EC 3.1.21.2; EC 3.1.21.3; EC 3.1.21.4; EC 3.1.21.5; EC 3.1.21.6 and EC 3.1.21.7)..

[0067] According to some embodiments, the DNase polypeptide is a DNase-like protein polypeptide. The term "DNase I-like protein" refers to an enzyme having an enzyme classification of EC 3.1.21.x, where "x" is a positive integer. As used herein, "DNase I-like proteins" are a subset of "DNases." The amino acid sequences of many DNases, and the coding sequences encoding such DNase proteins are well known in the art, available from public genomic databases such as GenBank, SwissProt, EMBL .

[0068] According to yet further embodiments of the invention, the human DNase I protein comprises an N-terminal glycine, and the human DNase I protein contiguously linked at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 12.

[0069] As mentioned, according to some embodiments of the invention, the human DNase I protein comprises the amino acid sequence as set forth in SEQ ID NO: 5. When plant-expressed human recombinant DNase I of the invention was subjected to controlled proteolytic digestion, mass spectrometry of the resulting oligopeptides revealed that the human DNase I polypeptide could be characterized by a group of overlapping peptide fragments, which together indicated that the full length recombinant human DNase I expressed by the plant cells was identical, in amino acid sequence, to that of native human DNase I, with the addition of a N-terminal glycine residue (see Example 2 and Tables V and Va, hereinbelow). Further analysis of the amino acid sequence confirmed the accuracy of the sequence and identity with the native human DNase I. Thus, according to some embodiments of some aspects of the invention, the inhalable pharmaceutical composition comprises a prh DNase I comprising a plurality of overlapping DNase I protein peptide fragments, the fragments having the amino acid sequence as set forth in SEQ ID NOs. 17-276 and 278-291. In some further embodiments, the prh DNase I has an additional N-terminal glycine residue, and in other embodiments, the prh DNase I is devoid of an N-terminal glycine residue. In some embodiments, the recombinant human DNase is characterized by consecutive overlapping peptide fragments, wherein peptide fragment SEQ ID NO: 17 represents an N-terminal fragment of the recombinant human DNase and peptide fragment SEQ ID NO: 276 represents a C-terminal fragment of the recombinant human DNase, and overlapping intermediary fragments include all or any of the peptide fragments represented by the amino acid sequences as set forth in SEQ ID NOs. 18-275 and 278-291.

[0070] According to some embodiments of some aspects of the invention, molecular mass of the plant-expressed recombinant human DNase polypeptide is similar to the molecular mass, as measured by PAGE and/or mass spectrometry, of recombinant human DNase I expressed in mammalian cells (Pulmozyme ®). Thus, in some embodiments of the invention, the plant-expressed recombinant human DNase polypeptide has a molecular mass of about 30 kD, as measured by SDS-PAGE, and about 32 kD, as measured by mass spectrometry. According to some embodiments, the electrophoretic mobility of the glycosylated plant expressed recombinant human DNase I protein is greater than that of recombinant human DNase I expressed in mammalian cells (Pulmozyme ®). In yet further embodiments, the plant-expressed recombinant human DNase polypeptide is a glycosylated protein, comprising a polypeptide moiety having a molecular mass of about 29 kD.

[0071] As shown in Examples 2 and 3, plant-expressed recombinant human DNase I is fully cross-reactive with anti-human recombinant DNase I anti-serum raised against mammalian-cell expressed recombinant human DNase I (e.g. Pulmozyme ®), for example, as detected by Western blotting of the gel- separated plant-expressed recombinant human DNase I (see Figure 4) and with ELISA (see Example 3). Thus, according to some embodiments, the plant-expressed recombinant human DNase I protein is immunoreactive with anti- human DNase I anti-serum. The anti-human DNase I antibody can be any of a polyclonal antibody, raised against human DNase I in a mammal, for example, rabbit, goat, horse, etc, or in other, non-mammalian vertebrates, for example, chicken IgY yolk antibody, can be an isolated antibody or can be in the form of anti-serum, or can also be a monoclonal anti-human DNase antibody.

[0072] As shown in Figures 5A-5D, isoelectric focusing of the plant-expressed recombinant human DNase I protein revealed that the plant-expressed recombinant human DNase I protein is characterized by two major isoforms with isoelectric points between 4.2 and 4.5 and a minor isoform with a pI between 4.2 and 3.5. Thus, in some embodiments the plant-expressed recombinant human DNase I protein is characterized by two major isoforms with isoelectric points between 4.2 and 4.5. In other embodiments, the plant-expressed recombinant human DNase I protein is further characterized by a minor isoform with a pI between 4.2 and 3.5.

[0073] According to some embodiments of some aspects of the invention, the prh DNase I is a plant cell-expressed human DNase protein secreted into the medium. Purification of the secreted plant cell-expressed human DNase protein from the medium yielded a composition comprising the prh human DNase protein, which was found to include less than 7% impurities (non-DNase I material), as monitored by RP-HPLC (see Figures 9A-9D). Thus, in some embodiments the prh human DNase I protein is a purified protein, characterized by a purity of at least 85%, at least 87%, at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%, at least 94%, at least 94.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, in a range of at least 95.0-99.8% or 100% purity. In some embodiments, purity of the plant-expressed recombinant human DNase I protein is measured by HPLC.

[0074] In some embodiments the plant-expressed recombinant human DNase I composition comprises impurities derived from the plant host cell, such as, but not limited to nucleic acids and polynucleotides, amino acids, oligopeptides and polypeptides, glycans and other carbohydrates, lipids and the like. In some embodiments the host-cell derived impurities comprise biologically active molecules, such as enzymes. In other embodiments, the plant-expressed recombinant human DNase I composition comprises plant beta-N-acetylhexosaminidase. Where the host cell is a tobacco cell, or tobacco cell line cell, the plant beta-N-acetylhexosaminidase is a tobacco beta-N-acetylhexosaminidase.

[0075] In further embodiments the plant beta-N-acetylhexosaminidase is inactivated plant beta-N-acetylhexosaminidase. Inactivation of plant beta-N-acetylhexosaminidase can be effected by physical means, chemical means or biochemical means. Physical inactivation can be performed by heating, freezing, desiccation, etc. Chemical inactivation can be performed by extremes of pH, chemical denaturation, addition or removal of side chains, glycans, amino acids, etc. Biochemical inactivation includes, but is not limited to inhibition by reversible or irreversible inhibitors. Exemplary beta-N-acetylhexosaminidase inhibitors include end-product inhibitors such as N-acetyl-D-glucosamine and beta-methyl-N-acetyl glucosamine, and selective inhibitors. It will be appreciated that preferred methods for inhibition and/or inactivation of the plant beta-N-acetylhexosaminidase are those which also effectively preserve the structural and functional integrity of the plant-expressed human DNase I enzyme.

[0076] In some embodiments the plant beta-N-acetylhexosaminidase is inactivated by heating the plant-expressed recombinant human DNase I composition. Suitable temperatures for plant beta-N-acetylhexosaminidase inhibition and/or activation include heating within a range of 37-60 °C for a period of 2 to 5, 10, 20, 30, 40, 50, 60 or more minutes. It will be appreciated that effective inhibition and/or inactivation of the plant beta-N-acetylhexosaminidase is achieved more rapidly at higher temperatures and more slowly at lower temperatures of the range. In some embodiments, the plant-expressed recombinant human DNase I composition is heated in the range of 45-55 °C for 2-10 minutes. In some embodiments, the inhibition/inactivation results in 20, 30, 40, 50, 60, 70, 80% or greater inactivation of the plant beta-N-acetylhexosaminidase.

[0077] According to another aspect of the invention, the human DNase I protein is contiguously linked at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide.

[0078] As used herein the term "contiguously linked at the N-terminal" refers to covalent attachment of the indicated peptide via a peptide bond to the N-terminal amino acid of the mature protein, for example, human DNase I protein. "Contiguously linked at the C-terminal" refers to covalent attachment of the indicated peptide via a peptide bond to the C-terminal amino acid of the mature protein, for example, human DNase I protein.

[0079] As used herein, the term "Arabidopsis ABPI endoplasmic reticulum targeting signal peptide" refers to the leader peptide sequence of the Arabidopsis thaliana auxin binding protein, which is capable of directing the expressed protein to the endoplasmic reticulum within the plant cell. In one embodiment, the Arabidopsis ABPI endoplasmic reticulum targeting signal peptide is a 33 amino acid polypeptide as set forth in SEQ ID NO: 4.

[0080] Thus, according to another aspect of the present invention, the human DNase I protein contiguously linked at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide and the human DNase I protein has an amino acid sequence as set forth in SEQ ID NO: 1.

[0081] According to some embodiments of the invention, the human DNase I protein is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 9. According to further embodiments of the invention, the Arabidopsis ABPI endoplasmic reticulum targeting signal peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 10. According to still further embodiments of the invention the human DNase I protein contiguously linked at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 12.

[0082] In order to express the polypeptide, the sequence encoding same is ligated into a "plant nucleic acid expression construct".

[0083] As used herein the term "plant nucleic acid expression construct" refers to a nucleic acid construct which includes the nucleic acid of some embodiments of the invention and at least one promoter for directing transcription of nucleic acid in a host plant cell. Further details of suitable transformation approaches are provided hereinbelow.

[0084] According to some embodiments of the invention, there is provided a nucleic acid expression construct comprising the nucleic acid sequence of the invention, and a promoter for directing transcription of the nucleic acid sequence in a plant host cell.

[0085] As used herein the term "nucleic acid sequence" refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

[0086] As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

[0087] As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

[0088] As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

[0089] According to some embodiments of the present invention, the nucleic acid sequences encoding the polypeptides of the present invention are optimized for expression in plants. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization. In one embodiment, the codon usage of the nucleic acid sequence encoding the human DNase I protein, the human DNase I protein contiguously linked at the N-terminal to an Arabidopsis ABPI endoplasmic reticulum targeting signal peptide, or any other human DNase I protein described herein is optimized for Nicotiana tabacuum or Nicotiana benthamiana.

[0090] The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU=n=1N[(Xn-Yn)/Yn]2/N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A table of codon usage from highly expressed genes of dicotyledonous plants has been compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

[0091] One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of Agrobiological Sciences) DNA bank in Japan (Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/). The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.

[0092] By using such codon optimization tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally-occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less-favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively affect mRNA stability or expression.

[0093] The desired encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically-favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the desired nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.

[0094] According to some embodiments of the invention, the nucleic acid is operably linked to the promoter sequence.

[0095] A coding nucleic acid sequence is "operably linked" to a regulatory sequence (e.g., promoter) if the regulatory sequence is capable of exerting a regulatory effect on (e.g. effect on the expression of) the coding sequence linked thereto.

[0096] Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. Preferably the promoter is a constitutive promoter, a tissue-specific, or an inducible promoter.

[0097] As used herein the phrase "plant-expressible" refers to a promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ. Such a promoter can be constitutive, i.e., capable of directing high level of gene expression in a plurality of tissues, tissue specific, i.e., capable of directing gene expression in a particular tissue or tissues, inducible, i.e., capable of directing gene expression under a stimulus, or chimeric, i.e., formed of portions of at least two different promoters.

[0098] Examples of preferred promoters useful for the methods of some embodiments of the invention are presented in Table I, II, III and IV.
Table I
Exemplary constitutive promoters for use in the performance of some embodiments of the invention
Gene SourceExpression PatternReference
Actin constitutive McElroy etal, Plant Cell, 2: 163-171, 1990
CAMV 35S constitutive Odell et al, Nature, 313: 810-812, 1985
CaMV 19S constitutive Nilsson et al., Physiol. Plant 100:456-462, 1997
GOS2 constitutive de Pater et al, Plant J Nov;2(6):837-44, 1992
ubiquitin constitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992
Rice cyclophilin constitutive Bucholz et al, Plant Mol Biol. 25(5):837-43, 1994
Maize H3 histone constitutive Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992
Actin 2 constitutive An et al, Plant J. 10(1);107-121, 1996
Table II
Exemplary seed-preferred promoters for use in the performance of some embodiments of the invention
Gene SourceExpression PatternReference
Seed specific genes seed Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, etal., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990.
 Brazil Nut albumin seed Pearson' et al., Plant Mol. Biol. 18: 235- 245, 1992.
legumin seed Ellis, et al.Plant Mol. Biol. 10: 203-214, 1988
Glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987
Zein seed Matzke et al Plant Mol Biol, 143).323-32 1990
napA seed Stalberg, et al, Planta 199: 515-519, 1996
 wheat LMW and HMW, glutenin-1 endosperm Mol Gen Genet 216:81-90, 1989; NAR 17:461-2,
Wheat SPA seed Albanietal, Plant Cell, 9: 171-184, 1997
wheat a, b and g gliadins endosperm EMBO3:1409-15, 1984
Barley ltrl promoter endosperm  
barley B1, C, D hordein endosperm Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996
Barley DOF endosperm Mena et al, The Plant Journal, 116(1): 53- 62, 1998
Biz2 endosperm EP99106056.7
Synthetic promoter endosperm Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998
rice prolamin NRP33 endosperm Wu et al, Plant Cell Physiology 39(8) 885- 889, 1998
rice -globulin Glb-1 endosperm Wu et al, Plant Cell Physiology 398) 885-889, 1998
rice OSH1 embryo Sato et al, Proc. Nati. Acad. Sci. USA, 93: 8117-8122
rice alpha-globulin REB/OHP-1 endosperm Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997
 rice ADP-glucose PP endosperm Trans Res 6:157-68, 1997
maize ESR gene family endosperm Plant J 12:235-46, 1997
sorghum gamma-kafirin endosperm PMB 32:1029-35, 1996
KNOX embryo Postma-Haarsma ef al, Plant Mol. Biol. 39:257-71, 1999
 rice oleosin Embryo and aleuton Wu et at, J. Biochem., 123:386, 1998
sunflower oleosin Seed (embryo and dry seed) Cummins, etal., Plant Mol. Biol. 19: 873- 876, 1992
Table III
Exemplary flower-specific promoters for use in the performance of the invention
Gene SourceExpression PatternReference
AtPRP4 flowers www.salus. medium.edu/m mg/tierney/html
chalene synthase (chsA) flowers Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990.
LAT52 anther Twell et al Mol. Gen Genet. 217:240-245 (1989)
apetala- 3 flowers  
Table IV
Alternative rice promoters for use in the performance of the invention
PRO #geneexpression
PR00001 Metallothionein Mte transfer layer of embryo + calli
PR00005 putative beta-amylase transfer layer of embryo
PR00009 Putative cellulose synthase Weak in roots
PR00012 lipase (putative)  
PR00014 Transferase (putative)  
PR00016 peptidyl prolyl cis-trans isomerase (putative)  
PR00019 unknown  
PR00020 prp protein (putative)  
PR00029 noduline (putative)  
PR00058 Proteinase inhibitor Rgpi9 seed
PR00061 beta expansine EXPB9 Weak in young flowers
PR00063 Structural protein young tissues+calli+embryo
PR00069 xylosidase (putative)  
PR00075 Prolamine 10Kda strong in endosperm
PR00076 allergen RA2 strong in endosperm
PR00077 prolamine RP7 strong in endosperm
PR00078 CBP80  
PR00079 starch branching enzyme I  
PR00080 Metallothioneine-like ML2 transfer layer of embryo + calli
PR00081 putative caffeoyl- CoA 3-0 methyltransferase shoot
PR00087 prolamine RM9 strong in endosperm
PR00090 prolamine RP6 strong in endosperm
PR00091 prolamine RP5 strong in endosperm
PR00092 allergen RA5  
PR00095 putative methionine aminopeptidase embryo
PR00098 ras-related GTP binding protein  
PR00104 beta expansine EXPB 1  
PR00105 Glycine rich protein  
PR00108 metallothionein like protein (putative)  
PR00110 RCc3 strong root  
PR00111 uclacyanin 3-like protein weak discrimination center / shoot meristem
PR00116 26S proteasome regulatory particle non-ATPase subunit 11 very weak meristem specific
PR00117 putative 40S ribosomal protein weak in endosperm
PR00122 chlorophyll a/lo-binding protein precursor (Cab27) very weak in shoot
PR00123 putative protochlorophyllide reductase Strong leaves
PR00126 metallothionein RiCMT strong discrimination center shoot meristem
PR00129 GOS2 Strong constitutive
PR00131 GOS9  
PR00133 chitinase Cht-3 very weak meristem specific
PR00135 alpha- globulin Strong in endosperm
PR00136 alanine aminotransferase Weak in endosperm
PR00138 Cyclin A2  
PR00139 Cyclin D2  
PR00140 Cyclin D3  
PR00141 Cyclophyllin 2 Shoot and seed
PR00146 sucrose synthase SS1 (barley) medium constitutive
PR00147 trypsin inhibitor ITR1 (barley) weak in endosperm
PR00149 ubiquitine 2 with intron strong constitutive
PR00151 WSI18 Embryo and stress
PR00156 HVA22 homologue (putative)  
PR00157 EL2  
PR00169 aquaporine medium constitutive in young plants
PR00170 High mobility group protein Strong constitutive
PR00171 reversibly glycosylated protein RGP1 weak constitutive
PR00173 cytosolic MDH shoot
PR00175 RAB21 Embryo and stress
PR00176 CDPK7  
PR00177 Cdc2-1 very weak in meristem
PR00197 sucrose synthase 3  
PR00198 OsVP1  
PR00200 OSH1 very weak in young plant meristem
PR00208 putative chlorophyllase  
PR00210 OsNRT1  
PRO0211 EXP3  
PRO0216 phosphate transporter OjPT1  
PRO0218 oleosin 18kd aleurone + embryo
PRO0219 ubiquitine 2 without intron  
PRO0220 RFL  
PRO0221 maize UBI delta intron not detected
PRO0223 glutelin-1  
PRO0224 fragment of prolamin RP6 promoter  
PRO0225 4xABRE  
PRO0226 glutelin OSGLUA3  
PRO0227 BLZ-2_short (barley)  
PR00228 BLZ-2_long (barley)  


[0099] The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication. According to some embodiments of the invention, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

[0100] The nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells. In stable transformation, the nucleic acid is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.

[0101] Thus, according to some aspects of the present invention, there is provided an isolated cell comprising the nucleic acid construct of the invention.

[0102] As used herein, the term "isolated cell" refers to a cell at least partially separated from the natural environment e.g., from a plant. In some embodiments, the isolated cell is a plant cell of a whole plant. In some embodiments, the isolated cell is a plant cell, for example, a plant cell in culture.

[0103] The term '"plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.

[0104] According to some embodiments of the invention, the plant or plant cell is a duckweed plant, cell or nodule. Duckweed (members of the monocotyledonous family Lemnaceae, or Lemna) plant or duckweed nodule cultures can be efficiently transformed with an expression cassette containing a nucleotide sequence of interest by any one of a number of methods including Agrobacterium-mediated gene transfer, ballistic bombardment, or electroporation. Methods for molecular engineering of duckweed cells and detailed description of duckweed expression systems useful for commercial production of polypeptides are known in the art (see, for example, US-Patent Nos. 6,040,498 and 6,815,184 to Stomp, et al, and 8,022,270 to Dickey et al..

[0105] According to some embodiments of the invention, the plant or plant cell used by the method of the invention is a crop plant or cell of a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton.

[0106] According to further embodiments the plant cells includes tobacco cells, Agrobacterium rihzogenes transformed root cell, celery cell, ginger cell, horseradish cell and carrot cells. In one embodiment the tobacco cells are from a tobacco cell line, such as, but not limited to Nicotiana tabacum L. cv Bright Yellow (BY-2) cells. The plant cells may be grown according to any type of suitable culturing method, including but not limited to, culture on a solid surface (such as a plastic culturing vessel or plate for example) or in suspension. It will be noted that some cells, such as the BY-2 and carrot cells can be cultured and grown in suspension. Suitable devices and methods for culturing plant cells in suspension are known in the art, for example, as described in International Patent Application PCT IL2008/000614. In yet another embodiment the cells are cells of whole tobacco plants or plant tissues, including, but not limited to Nicotiana benthamiana.

[0107] There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276).

[0108] The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
  1. (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
  2. (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.


[0109] The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

[0110] There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

[0111] Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

[0112] Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

[0113] Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.

[0114] According to some embodiments of the invention, the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.

[0115] Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

[0116] Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.

[0117] According to some embodiments of the invention, the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

[0118] Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.

[0119] Construction of plant RNA viruses for the introduction and expression of non-viral nucleic acid sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990) 269:73-76; and U.S. Pat. No. 5,316,931.

[0120] When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.

[0121] In one embodiment, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

[0122] In a second embodiment, a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.

[0123] In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

[0124] In a fourth embodiment, a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

[0125] The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.

[0126] Techniques for inoculation of viruses to plants may be found in Foster and Taylor, eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and Koprowski, eds. "Methods in Virology" 7 vols, Academic Press, New York 1967-1984; Hill, S.A. "Methods in Plant Virology", Blackwell, Oxford, 1984; Walkey, D.G.A. "Applied Plant Virology", Wiley, New York, 1985; and Kado and Agrawa, eds. "Principles and Techniques in Plant Virology", Van Nostrand-Reinhold, New York.

[0127] In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.

[0128] A technique for introducing exogenous nucleic acid sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the nucleic acid sequence includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050 and 5,693,507. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.

[0129] According to some embodiments of the invention, the method further comprises growing the plant cell expressing the nucleic acid. The plant cells can be any plant cells desired. The plant cells can be cultured cells, cells in cultured tissue or cultured organs, or cells in a plant. In some embodiments, the plant cells are cultured cells, or cells in cultured tissue or cultured organs. In yet further embodiments, the plant cells are any type of plant that is used in gene transference. The plant cell can be grown as part of a whole plant, or, alternatively, in plant cell culture.

[0130] According to some aspects of the invention, the plant cells are grown in a plant cell suspension culture. As used herein, the term "suspension culture" refers to the growth of cells separate from the organism. Suspension culture can be facilitated via use of a liquid medium (a "suspension medium"). Suspension culture can refer to the growth of cells in liquid nutrient media. Methods and devices suitable for growing plant cells of the invention in plant cell suspension culture are described in detail in, for example, PCT WO2008/135991, US Patent No. 6,391,683, US Patent Application No. 10/784,295; International Patent Publications PCT Nos. WO2004/091475, WO2005/080544 and WO2006/040761.

[0131] Thus, the invention encompasses plants or plant cultures expressing the nucleic acid sequences, so as to produce the recombinant human DNase I protein of the invention. Once expressed within the plant cell or the entire plant, the level of the human DNase I protein encoded by the nucleic acid sequence can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the human DNase I protein, Enzyme-Linked Immuno Sorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.

[0132] Methods of determining the level in the plant of the RNA transcribed from the nucleic acid sequence are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.

[0133] According to some embodiments of the invention, the expressed recombinant human DNase I protein of the present invention is glycosylated in the plant cell, resulting in a recombinant human DNase I protein having high mannose glycosylation (e.g. exposed mannose sugar residues), and plant specific glycan residues. Thus, according to some embodiments of the invention, the cells expressing the expression vector of the invention produce a human DNase I protein having at least one, optionally at least two, optionally at least three or optionally at least four or more exposed mannose residues. In other embodiments the cells expressing the expression vector of the invention produce a human DNase I protein having at least one, optionally at least two, optionally at least three or optionally at least four or more core xylose residues. In yet other embodiments the cells expressing the expression vector of the invention produce a human DNase I protein having at least one, optionally at least two, optionally at least three or optionally at least four or more core α-(1,3) fucose residues. In one embodiment the cells expressing the expression vector of the invention produce a human DNase I protein having at least one exposed mannose residue, at least one core xylose residue and at least one α-(1,3) fucose residue. In yet further embodiments, the cells expressing the expression vector of the invention produce a human DNase I protein having at least one, at least two, at least 3 or more terminal N-acetyl glucosamine substitutions on the outer mannose sugars.

[0134] Glycan analysis of human DNase I protein expressed in plant cells indicated that the prh DNase I lacks sialic acid residues. Thus, in yet other embodiments, the cells expressing the expression vector of the invention produce a recombinant human DNase I protein devoid of sialic acid residues.

[0135] The human DNase I protein produced by cells expressing the expression vector of the invention was shown to have DNase I catalytic activity similar or superior to that of a native human or mammalian-cell produced DNase I enzyme.

[0136] As shown in Example 3 herein, when enzyme kinetics of the plant-expressed recombinant human DNase I protein were measured (see Table VI), greater substrate affinity (KM) and higher reaction velocity (Vmax) were observed for the plant-expressed recombinant human DNase I protein, as compared to those of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). Thus, according to some embodiments the plant expressed recombinant human DNase I is biologically active. In some embodiments, the biological activity is endonuclease catalytic activity. In yet other embodiments, the biological activity of the plant-expressed recombinant human DNase I protein is characterized by greater substrate affinity (KM) and higher reaction velocity (Vmax), as compared to those of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). Methods for measuring biological activity of DNase I are well known in the art, and include, inter alia, catalytic activity (e.g. methyl green assay, DNaseAlert™-based assay, fluorescence-based assay, as described herewith), immuno-reactivity with anti-DNase I antibody, Kunitz-based hyperchromicity assay, assays measuring proton release upon DNA hydrolysis, which can be monitored by using chromophoric H+ ion indicators, and the like. In still other embodiments, the biological activity of the plant-expressed recombinant human DNase I protein is characterized by specific activity (units DNase I catalytic activity per mg enzyme protein) greater than that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). In some embodiments, the specific activity of plant-expressed recombinant human DNase I protein is in a range of about 1-10, about 1-1.1, about 1-1.25, about 1-1.5, about 1-1.75, about 1-2, about 1-2.25, about 1-2.5, about 1-2.75, about 1-3.0, about 1-3.25, about 1-3.5, about 1-3.75, about 1-4.0, about 2.5-3.0, about 2.5-3.1, about 2.5-3.2, about 2.5-3.3, about 2.5-3.4, about 2.5-3.5, about 1-4.5, about 1-5.0 or more fold that of the specific activity (e.g. U/mg) of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). In some embodiments, the specific activity of plant-expressed recombinant human DNase I protein is about 3.0 to 3.5 fold that of the specific activity (e.g. U/mg) of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®).

[0137] As shown in Example 4 herein, when enzyme activity of DNase I protein was measured (see Table VIII) in the presence of increasing concentrations of actin, greater resistance of the plant-expressed recombinant human DNase I to inhibition of catalytic activity by actin was observed, compared to that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). Thus, according to some embodiments the plant expressed recombinant human DNase I has greater resistance to actin inhibition of DNase I catalytic activity when compared to that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). In some embodiments, the inhibition of DNase I catalytic activity is expressed as half-maximal inhibitory concentration of actin (IC50, µg actin/ml), using, for example, the methyl green DNase I assay. Thus, in some embodiments, the IC50 actin concentration for inhibition of the plant expressed recombinant human DNase I is at least 1 to 10, 2 to 6, 1 to 1.1, 1 to 1.2, 1 to 1.25, 1 to 1.3, 1 to 1.4, 1 to 1.5, 1 to 1.6, 1 to 1.7, 1 to 1.8, 1 to 1.9, 1 to 2, 1 to 2.1, 1- 2.2, 1 to 2.25, 1 to 2.5, 1 to 2.75, 1 to 3.0, 1 to 3.25, 1 to 3.5, 1 to 3.75, 1 to 4.0, 1 to 4.5 to 1 to 5.0 or more, or a range of about 1.5 to 2.5 that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®). In some embodiments, the IC50 actin concentration for inhibition of the plant expressed recombinant human DNase I activity is about 2.0 to 2.2 fold that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®).

[0138] As shown in Example 5, plant expressed recombinant human DNase I effectively reduces the rheological properties of sputum. When measured in an in-vitro assay, incubation of the plant expressed recombinant human DNase I with samples of CF sputum results in reduction of the viscous modulus (as expressed by the loss modulus, G") and reduction of the elasticity (as expressed by the storage modulus, G') of the sputum sample (see Figures 12A-12D, 13A-13D, 14A-14B, 16 and 17). When compared to the reduction of rheological parameters of sputum samples incubated with mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®), the plant expressed recombinant human DNase I displayed greater efficacy in reducing viscosity and elasticity than that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®) (Figures 12 and 13). Thus, according to some embodiments of the invention, the plant expressed recombinant human DNase I reduces viscous modulus and/or elastic modulus of sputum. In other embodiments, reduction in viscous and elastic modulus is expressed as G" and G', respectively. In yet another embodiment, reduction of viscous and/or elastic modulus of sputum by the plant expressed recombinant human DNase I is greater than that of mammalian cell-expressed recombinant human DNase I (e.g. Pulmozyme ®) measured with the same assay technique. Rheological properties of sputum can be measured using methods assessing dynamic oscillation, creep recovery, stretch relaxation and the like, using a rheometer, extensograph, alveograph, etc. Measurement of viscous and elastic modulus by dynamic oscillation on a rheometer is detailed herewith. In other embodiments, the rheological properties of sputum are assayed using stress sweep measurements, and can be characterized by the cross-over points of elastic and viscous stress values.

[0139] It will be appreciated that the biological properties of the prh DNase I of the invention (i.e. endonuclease, reduced actin inhibition) make the prh DNase I of the invention an attractive candidate for lysis of DNA in any composition, for example an aqueous or semi-aqueous composition, such as but not limited to mucus, sputum, sperm, or other secretions, in which reduction of the DNA content, and subsequent reduction of rheological properties is desirable. Such increased rheological properties of secretions are often the result of viral or microbial growth and cytotoxic response of the host's cellular immunity.

[0140] Thus, in some embodiments, the plant expressed human recombinant DNase I of the invention is biologically active, having catalytic activity, enzyme kinetics and specific activity comparable or superior to that of mammalian cell-expressed recombinant human DNase I, and effective in reducing rheological properties of CF sputum. Thus, according to some aspects of some embodiments of the invention, there is provided a method for reducing a rheological property of a secretion containing DNA, the method comprising exposing the secretion to an effective concentration of the plant expressed human recombinant DNase I of the invention, thereby reducing a rheological property of the secretion. In some embodiments, exposing the secretion reduces the elastic modulus of the secretion. In other embodiments, exposing the mucus reduces the viscous modulus of the secretion. In some embodiments the secretion is a human secretion, selected from the group consisting of sputum, mucus and sperm. In another embodiment, the secretion is sputum or mucus. In yet another embodiment, the secretion is associated with a disease or condition resulting in increased amounts of DNA in the secretion, as compared to the DNA content of similar secretion from a healthy individual.

[0141] It will be appreciated that prh DNase I can be used to reduce the DNA content of biological fluids other than secretions, for example, blood, plasma, lymph, CSF and the like, or in the local environment of an internal organ or tissue of an organism, for example, animal and/or human subject. In some embodiments, administration of prhDNase I to the organism results in increased endonuclease activity in blood, for example, in circulating blood, or in a tissue of the organism. In yet other embodiments, the DNA content of the biological fluid, for example, blood is associated with a disease or condition resulting in increased amounts of DNA in the secretion, as compared to the DNA content of similar secretion from a healthy individual.

[0142] Thus, the human DNase I protein expressed in plant cells according to the invention can be used to produce a pharmaceutical composition. The pharmaceutical composition can be used for treatment or prevention of any condition or disease by any route of administration. According to some aspects of the invention, the pharmaceutical composition can be used for treatment or prevention of mucus-associated conditions in a subject in need thereof. According to other aspects of the invention, the pharmaceutical composition can be used for reducing the amount of DNA in a pulmonary secretion or fluid in a subject in need thereof. In some embodiments, the mucus associated conditions comprise respiratory or pulmonary disease or conditions. In further embodiments, methods for treatment or prevention of respiratory or pulmonary conditions comprise administering the pharmaceutical composition(s) of the invention by inhalation.

[0143] Respiratory conditions or diseases which can be treated by administration of plant-cell expressed recombinant human DNase I protein of the invention include conditions associated with accumulation of mucus, for example, in the airways. Such conditions include, but are not limited to acute or chronic bronchopulmonary disease, atelectasis due to tracheal or bronchial impaction and complications of tracheostomy chronic bronchitis, asthmatic bronchitis, cystic fibrosis, pneumonia, allergic diseases such as allergic asthma, non-allergic asthma, systemic lupus erythematosus, Sjogren's syndrome, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold.

[0144] Thus, according to another aspect of the present invention there is provided a pharmaceutical composition which includes, as an active ingredient thereof, a plant-expressed human DNase I protein (prh DNase I), and a pharmaceutical acceptable carrier. In some embodiments of the present invention, the human DNase I protein has an amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments of the present invention, the human DNase I protein has an N-terminal Glycine residue (SEQ ID NO: 5). In yet other embodiments, the human DNase protein is contiguously linked at the N-terminal to an endoplasmic reticulum signal peptide SEQ ID NO: 1. In still other embodiments the human DNase I protein is linked at the N-terminal to an ER signal peptide and has an N-terminal Glycine residue.

[0145] In further embodiments, biological activity of the plant expressed recombinant human DNase I of the invention is enhanced by the presence of an additional pharmacological agent, for example, an agent which reduces actin inhibition of DNase activity, such as one or more inorganic salt selected from potassium, magnesium, calcium, zinc, lithium, manganese, cadmium, nickel, cobalt, ammonium, polyamine and macrocyclic polyammonium salts. Agents suitable for combination with the plant expressed human recombinant DNase I of the invention, for therapeutic applications such as treatment of pulmonary conditions (e.g. CF) are described in detail in US 7,432,308 to Demeester et al.

[0146] In some embodiments, combination of the plant expressed recombinant human DNase I with an additional pharmaceutical agent results in improvement, and optionally synergic improvement in reduction of rheological properties (e.g. viscous modulus and/or elastic modulus) of sputum. In some embodiments, the additional agent is magnesium chloride or magnesium sulfate.

[0147] In some embodiments of the invention, the pharmaceutical composition is combined with, or administered along with an additional pharmaceutical agent, the additional pharmaceutical agent including, but not limited to one or more other pharmacologic agents used to treat the conditions listed above, such as antibiotics, bronchodilators, anti-inflammatory agents, mucolytics (e.g. n-acetyl-cysteine), actin binding or actin severing proteins [e.g., gelsolin; Matsudaira et al., Cell 54:139-140 (1988); Stossel, et al., PCT Patent Publication No. WO 94/22465], protease inhibitors, or gene therapy product [e.g., comprising the cystic fibrosis transmembrane conductance regulator (CFTR) gene, Riordan, et al., Science 245:1066-1073 (1989)]. Additional pharmaceutical agents can be administered prior to, along with, subsequent to or in any other temporal combination with the pharmaceutical composition of the invention. Regimen for combination of the pharmaceutical composition of the invention with additional agents can be formulated according to parameters such as specific conditions or diseases, health status of the subject, methods and dose of administration, and the like. Determination of such combination regimen can be done, for example, by professionals such as attending physicians, hospital staff, and also according to predetermined protocols.

[0148] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0149] Herein the term "active ingredient" refers to the recombinant human DNase I protein accountable for the biological effect.

[0150] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0151] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0152] In yet other embodiments the pharmaceutical composition is combined with, or administered along with a mixture of solid particulate ingredients within an oil carrier. Preferably the compositions are anhydrous. Specifically, the pharmaceutical compositions comprise a mixture of pharmacologically inert silica nanoparticles having a hydrophobic surface, a polysaccharide, where the particulate mixture is suspended or embedded in an oil. Detailed description of such a mixture is provided, for example, in US Patent Application 2010/297245 to Vol, et al.

[0153] Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

[0154] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0155] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

[0156] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient or other subject in need thereof.

[0157] For treatment and/or prevention of respiratory and/or pulmonary disorders, the recombinant human DNase I of the invention can be administered directly into the airways by pulmonary administration.

[0158] As used herein, the term "subject in need thereof' refers to a subject diagnosed with or exhibiting one or more conditions associated with a disease or condition treatable by administration of DNase I, a subject who has been diagnosed with or exhibited one or more conditions treatable by administration of DNase I in the past, or a subject who has been deemed at risk of developing one or more conditions associated with a disease or condition treatable by administration of DNase I in the future due to hereditary or environmental factors. In certain embodiments of the invention, the subject in need thereof is suffering from a disease or condition such as, but not limited to respiratory and/or pulmonary disease or condition, male infertility, viral, bacterial, fungal or protozoan infection, a uterine disorder, an endometrial disorder or condition, cancer, primary cancer and/or metastatic cancer.

[0159] In some embodiments, a subject in need thereof refers to a subject with a pulmonary condition having clinically abnormal spirometry values. Examples of spirometry parameters which can indicate the need of a subject include, but are not restricted to forced expiration volume1 (FEV1), forced vital capacity (FVC), forced expiratory flow (FEF25-75) and the like. In some embodiments of the invention administration of the prhDNase to the subject results in an improvement in one or more of the spirometric parameters.

Pulmonary administration of DNase



[0160] Pulmonary administration may be accomplished by suitable means known to those in the art. Pulmonary administration of DNase requires dispensing of the biologically active substance from a delivery device into the oral cavity of a subject during inhalation. For purposes of the present invention, compositions comprising DNase are administered via inhalation of an aerosol or other suitable preparation that is obtained from an aqueous or nonaqueous solution or suspension form, or a solid or dry powder form of the pharmaceutical composition, depending upon the delivery device used. Such delivery devices are well known in the art and include, but are not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, or any other appropriate delivery mechanisms that allow for dispensing of a pharmaceutical composition as an aqueous or nonaqueous solution or suspension or as a solid or dry powder form. Methods for delivering DNase, including DNase I, to a subject via pulmonary administration, including directed delivery to the central and/or peripheral lung region(s), include, but are not limited to, a dry powder inhaler (DPI), a metered dose inhaler (MDI) device, and a nebulizer.

Nebulizer/Liquid Inhaler



[0161] In one embodiment, the DNase, including an enzymatically active portion thereof, is delivered to a subject using a nebulizer or liquid inhaler. Generally, nebulizers use compressed air to deliver medicine as wet aerosol or mist for inhalation, and, therefore, require that the drug be soluble in water. Nebulizer devices can deliver relatively large doses in comparison to MDI (metered dose inhaler) or DPI (dry powder inhaler) devices, and are especially effective for delivery to the deep lung (peripheral lung region). No propellants are required for nebulizers, which includes jet nebulizers (air-jet nebulizers and liquid-jet nebulizers) and ultrasonic nebulizers. Examples of nebulizers include AkitaTM (Activaero GmbH) (see US Pat. No. 7,766,012, EP1258264), a table top nebulizer inhalation system based on Pari's LC Star that provides full control over patient's breathing pattern and the portable Aeroneb® Go/Pro/Lab nebulizers (AeroGen). The Aeroneb® nebulizer is based on OnQTM technology, i.e., an electronic micropump surrounded by a vibrational element and adaptable to the needs of a broad range of patients, including children and the elderly; single or multi-patient use.

[0162] The portable AerocurrentTM (AerovertRx corp) may also be used in the methods and compositions of the invention (see WO2006006963).

[0163] Staccato™ (Alexza Pharma) may also be used in the methods and compositions of the invention (see WO03095012). The key to Staccato™ technology is vaporization of a drug without thermal degradation. AERx® (Aradigm), a hand held battery operated device, may also be used in the methods and compositions of the invention (see WO9848873, U.S. Pat. No. 5,469,750, U.S. Pat. No. 5,509,404, U.S. Pat. No. 5,522,385, U.S. Pat. No. 5,694,919, U.S. Pat. No. 5,735,263, U.S. Pat. No. 5,855,564). Another example of a nebulizer device which may also be used in the methods and compositions of the invention includes Respimat® (Boehringer), a multidose reservoir system. The DNase may also be delivered using the Collegium NebulizerTM (Collegium Pharma). Another example of a nebulizer device which may also be used in the methods and compositions of the invention includes the Inspiration® 626 (Respironics), a compressor based nebulizer for home care delivering a particle size between 0.5 to 5 microns, Adaptive Aerosol Delivery® technology (Respironics), which delivers precise and reproducible inhaled drug doses.

[0164] Adaptive Aerosol Delivery (AAD)® systems incorporate electronics and sensors within the handpiece to monitor the patient's breathing pattern by detecting pressure changes during inspiration and expiration, allowing the patient to take breaks in therapy without medication waste. Examples of AAD® system nebulizers include the HaloLite® AAD®, ProDose® AAD®, and I-Neb® AAD®. The HaloLite® Adaptive Aerosol Delivery (AAD)® (Respironics) is a pneumatic aerosolisation system powered by a portable compressor (see EP 0910421).

[0165] The ProDos AAD® (Respironics) is a pneumatic aerosol system powered by a portable compressor, controlled by "ProDose DiscTM" system. (Respironics). (see EP1245244). Promixin® can be delivered via Prodose AAD® for management of pseudomonas aeruginosa lung infections, particularly in cystic fibrosis. Promixin® is supplied as a powder for nebulization that is reconstituted prior to use. The I-neb AAD® is a handheld, miniaturized AAD® system without the need for a separate compressor ("I-Neb"), based upon a combination of electronic mesh-based aerosolisation technology (Omron) and AAD® technology. I-neb AAD® has been used for delivery of Ventavis® (iloprost) (CoTherix/Schering AG).

[0166] Another example of a nebulizer which may be used in the methods and compositions of the invention is AriaTM (Chrysalis). Aria is based on a capillary aerosol generation system with MMAD ranging from 0.5-2.0 µm.

[0167] In another embodiment, the TouchSpray™ nebulizer (Odem), which uses a perforate membrane, which vibrates at ultrasonic frequencies, in contact with the reservoir fluid, to generate the aerosol cloud (see U.S. Pat. No. 6,659,364) may be used to deliver DNase in accordance with the invention. Additional nebulizers which may be used in the invention include nebulizers which are portable units which maximize aerosol output when the patient inhales and minimize aerosol output when the patient exhales using two oneway valves (see PARI nebulizers (PARI GmbH), which may be designed for specific patient populations, such a patients less than three years of age (PARI BABYTM) and nebulizers for older patients (PARI LC PLUS® and PARI LC STAR®).

[0168] An additional nebulizer which may be used in the invention is the e-Flow® nebulizer (PARI GmbH) which uses vibrating membrane technology to aerosolize the drug solution, as well as the suspensions or colloidal dispersions (TouchSpray™; ODEM (United Kingdom)), as described in U.S. Pat. No. 6,962,151. Additional nebulizers which may be used in the invention include the Hudson T-Updraft I or II nebulizer (Pulmo-Aide compressor), Marquest Acorn I or II nebulizer (Pulmo-Aide compressor), Durable Sidestream (Portaneb compressor), the Microair® electronic nebulizer (Omron)(see US Pat. No. 6,901,926) and a MysticTM nebulizer (Ventaira)(see U.S. Pat. No. 6,397,838). The MysticTM device is breath activated, and has been used with Corus 1030TM (lidocaine HC1), Resmycin® (doxorubicin hydrochloride), Acuair (fluticasone propionate), NCE with ViroPharm, and NCE with Pfizer. Thus, in one embodiment, the invention provides a container for use with a nebulizer device for pulmonary administration of DNase to a subject, the container comprising a propellant-free inhalable solution or suspension comprising the DNase.

[0169] The DNase may be administered to a subject via inhalation in accordance with a dosing regimen designed to achieve a therapeutic effect. In one embodiment, a multiple dosing regimen may be used to treat disorders in which DNase is beneficial using the methods described herein. Multiple variable dose methods of treatment can also be used to treat disorders in which DNase is beneficial.

Pharmaceutical Compositions



[0170] DNase for use in the methods of the invention, can be incorporated into pharmaceutical compositions suitable for pulmonary administration to a subject. The compositions for use in the methods and compositions of the invention may be in a variety of forms according to the mode of inhalation and therapeutic application. In one embodiment, the invention provides a pharmaceutical composition comprising a DNase and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for inhalation by a subject. Thus, the DNase is formulated in a pharmaceutical composition suitable for inhalation. Examples of pharmaceutical compositions which are suitable for inhalation include, but are not limited to a propellant-containing aerosol, and a propellant-free inhalable solution or suspension. Such pharmaceutical compositions may be administered according to the devices described above. For example, a propellant-free inhalable solution comprising the DNase may be administered to the subject via a nebulizer. Other suitable preparations include, but are not limited to, mist, vapor, or spray preparations so long as the particles comprising the protein composition are delivered in a size range consistent with that described for the delivery device, e.g., a dry powder form of the pharmaceutical composition.

[0171] Where a liquid solution or suspension is used in the delivery device, a nebulizer, a metered dose inhaler, or other suitable delivery device delivers, in a single or multiple fractional dose, by pulmonary inhalation a pharmaceutically effective amount of the composition to the subject's lungs as droplets having the same particle size range noted above for the dry powder form. Methods for preparing and using formulations suitable for use as liquid or suspension are known in the art, for example, the oil based matrix taught in PCT WO 2011/004476.

[0172] Where the liquid pharmaceutical composition is lyophilized prior to use in the delivery methods of the invention, the lyophilized composition may be milled to obtain the finely divided dry powder consisting of particles within the desired size range noted above. Where spray-drying is used to obtain a dry powder form of the liquid pharmaceutical composition, the process is carried out under conditions that result in a substantially amorphous finely divided dry powder consisting of particles within the desired size range noted above. Similarly, if the starting pharmaceutical composition is already in a lyophilized form, the composition can be milled to obtain the dry powder form for subsequent preparation as an aerosol or other preparation suitable for pulmonary inhalation. Where the starting pharmaceutical composition is in its spray-dried form, the composition has preferably been prepared such that it is already in a dry powder form having the appropriate particle size for dispensing as an aqueous or nonaqueous solution or suspension in accordance with the pulmonary administration methods of the invention. For methods of preparing dry powder forms of pharmaceutical compositions, see, for example, WO 96/32149, WO 97/41833, WO 98/29096, and U.S. Pat. Nos. 5,976,574, 5,985,248, and 6,001,336.

[0173] The resulting dry powder form of the composition is then placed within an appropriate delivery device for subsequent preparation as an aerosol or other suitable preparation that is delivered to the subject via pulmonary inhalation. Where the dry powder form of the pharmaceutical composition is to be prepared and dispensed as an aqueous or non-aqueous solution or suspension, a metered-dose inhaler, or other appropriate delivery device is used.

[0174] The dry powder form of the pharmaceutical composition comprising a DNase may be reconstituted to an aqueous solution for subsequent delivery as an aqueous solution aerosol using a nebulizer, a metered dose inhaler, or other suitable delivery device. In the case of a nebulizer, the aqueous solution held within a fluid reservoir is converted into an aqueous spray, only a small portion of which leaves the nebulizer for delivery to the subject at any given time. The remaining spray drains back into a fluid reservoir within the nebulizer, where it is aerosolized again into an aqueous spray. This process is repeated until the fluid reservoir is completely dispensed or until administration of the aerosolized spray is terminated. Examples of nebulizers are described above.

[0175] The composition of the invention can be formulated with addition ingredients. These can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel™, or corn starch; a lubricant such as magnesium stearate or Sterotes™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

[0176] The stabilized lyophilized or spray-dried compositions may be formulated using a buffering agent, which maintains the pH of the pharmaceutical composition within an acceptable range when in a liquid phase, such as during the formulation process or following reconstitution of the dried form of the composition. In some embodiments the pH is in the range of about pH 4.0 to about pH 8.5, about pH 4.5 to about pH 7.5, about pH 5.0 to about pH 6.5, about pH 5.6 to about pH 6.3, and about pH 5.7 to about pH 6.2. Suitable pH's include about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.8, about 7.9, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0, in the range of 3.5-9.0, 4.0-8.0, 4.5.0-7.5, 5.0-7.5, 5.5-7.0 and 6.0-7.0. In one particular embodiment, the pH is about 7.0 to 8.2. Suitable buffering agents include, but are not limited to, citrate buffer, phosphate buffer, succinate buffer, more particularly a sodium citrate/citric acid. Alternatively imidazole or histidine or other base/acid that maintains pH in the range of about pH 4.0 to about 8.5 can be used. Buffers are chosen such that they are compatible with the drying process and do not affect the quality, purity, potency, and stability of the protein during processing and upon storage.

[0177] Any of the pharmaceutical compositions comprising DNase contemplated for use in the methods of the invention may be formulated with at least one surfactant. For pulmonary intracellular administration of the DNase, the surfactant can be in an amount sufficient to enhance absorption of the inhaled particles comprising DNase to obtain an absorbable composition for use in pulmonary inhalation in accordance with the methods described herein. Any surfactant that enhances absorption of a pharmaceutical composition comprising DNase thereof in the manner disclosed herein may be used to obtain these absorbable protein-containing pharmaceutical compositions. Surfactants suitable for use in enhancing absorption of the inhaled DNase include, but are not limited to, polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters such as Poloxamer 188; polyoxyethylene alcohols such as Brij 35; a mixture of polysorbate surfactants with phospholipids such as phosphatidylcholine and derivatives (dipalmitoyl, dioleoyl, dimyristyl, or mixed derivatives such as 1-palmitoyl, 2-olcoyl, etc.), dimyristolglycerol and other members of the phospholipid glycerol series; lysophosphatidylcholine and derivatives thereof; mixtures of polysorbates with lysolecithin or cholesterol; a mixture of polysorbate surfactants with sorbitan surfactants (such as sorbitan monoleate, dioleate, trioleate or others from this class); poloxamer surfactants; bile salts and their derivatives such as sodium cholate, sodium deoxycholate, sodium glycodeoxycholate, sodium taurocholate, etc.; mixed micelles of DNase with bile salts and phospholipids; Brij surfactants (such as Brij 35-PEG923) lauryl alcohol, etc.). The amount of surfactant to be added is in the range of about 0.005% to about 1.0% (w/v), preferably about 0.005% to about 0.5%, more preferably about 0.01% to about 0.4%, even more preferably about 0.03% to about 0.3%, most preferably about 0.05% to about 0.2%.

[0178] The pharmaceutical composition of the invention may include a suitable dosage according to the disorder being treated. In one embodiment, the pharmaceutical composition of the invention comprises a dose of about 0.01 mg to 10 mg of DNase or enzymatically active portion thereof. Alternatively, the pharmaceutical composition of the invention comprises a dose of about 0.1 mg to 5 mg; about 1 mg to 5 mg; about 2.5 mg to 5 mg, about 2.0 to 4.5 mg, about 2.2 to 4.0 mg, about 2.0 to 3.0 mg, about 2.2 to 3.0 mg, about 2.3 to 3.0 mg, about 2.4 to 2.8 mg, about 2.4 to 2.6 mg; or about 2.5 mg of the DNase or enzymatically active portion thereof. In another embodiment, the pharmaceutical composition comprises a dose over 10 mg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0179] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile inhalable solutions can be prepared by incorporating the active compound (i.e., rhDNase, DNase I or other DNase) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged action of inhalable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0180] In one embodiment, the DNase or active portion for use in the methods of the invention is incorporated into a pharmaceutical formulation as described herein. Supplementary active compounds can also be incorporated into the compositions for pulmonary delivery. In certain embodiments, a DNase or active portion for use in the methods of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents mentioned hereinabove. For example, DNase I or active portion of the invention may be coformulated and/or coadministered with one or more additional compositions that reduce actin inhibition (e.g. magnesium or potassium salts), and/or one or more chemical agents that inhibit mucus production (such as antiinflammatory agents, bronchodilators and/or mucus secretion blockers, as described in US 7,763,610) or any combination thereof. Furthermore, the DNase of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible side effects, complications or low level of response by the patient associated with the various monotherapies.

[0181] The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a DNase protein or active portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the DNase may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the DNase or active portion thereof to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the DNase are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0182] The invention also pertains to packaged pharmaceutical compositions or kits for pulmonary administration of a DNase, e.g., DNase I. In one embodiment of the invention, the kit comprises a DNase, such as DNase I, and instructions for pulmonary administration of the DNase, wherein the DNase is in a formulation suitable for inhalation. The instructions may describe when, e.g., at day 1, day 4, week 0, week 2, week 4, etc., the different doses of DNase shall be administered via inhalation to a subject for treatment.

[0183] Another aspect of the invention pertains to kits containing a pharmaceutical composition comprising a DNase, such as DNase I, and a pharmaceutically acceptable carrier, and one or more pharmaceutical compositions each comprising an additional therapeutic agent, and a pharmaceutically acceptable carrier.

[0184] The package or kit alternatively can contain the DNase and it can be promoted for use, either within the package or through accompanying information, for the uses or treatment of the disorders described herein. The packaged pharmaceuticals or kits further can include a second agent (as described herein) packaged with or copromoted with instructions for using the second agent with a first agent (as described herein).

[0185] The term "tissue" refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

[0186] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0187] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0188] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, for example, the surfactants described hereinabove.

[0189] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0190] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0191] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0192] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0193] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0194] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0195] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use, as detailed hereinabove.

[0196] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0197] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (recombinant human DNase I) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cystic fibrosis) or prolong the survival of the subject being treated.

[0198] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0199] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0200] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0201] Dosage amount and interval may be adjusted individually, for example, to provide serum and cell levels of the active ingredient which are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0202] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0203] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0204] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0205] The pharmaceutical composition can be used for treatment or prevention of a mucus-related respiratory condition in a subject in need thereof. Thus, according to another aspect of the present invention there is provided a method for treating or preventing a respiratory condition such as cystic fibrosis (CF) or COPD in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition which includes, as an active ingredient thereof, a human DNase I protein, and a pharmaceutical acceptable carrier. In some embodiments of the present invention, the human DNase I protein has an N-terminal Glycine residue. In other embodiments, the human DNase I protein has an amino acid sequence as set forth in SEQ ID NO: 5.

[0206] In some embodiments of the invention, treatment of cystic fibrosis is effected by pulmonary administration of the prh DNase I, the effective dosage being in a range of 0.1 to 50 mg DNase I protein (as active ingredient) per dose, 0.1 to 50 mg DNase I protein (as active ingredient) per dose, 0.5 to 25 mg DNase I protein (as active ingredient) per dose, 1.0 to 20 mg DNase I protein (as active ingredient) per dose, 1.5 to 15 mg DNase I protein (as active ingredient) per dose, 2.0 to 10 mg DNase I protein (as active ingredient) per dose, 2.5 to 7.5 mg DNase I protein (as active ingredient) per dose, 2.75 to 5 mg DNase I protein (as active ingredient) per dose or from 2.0 to 3.0 mg DNase I protein (as active ingredient) per dose. In some embodiments the effective dose of prh DNase I is 2.0- 3.0 mg DNase I protein (as active ingredient) per dose, 2.1- 2.9 mg DNase I protein (as active ingredient) per dose, 2.2- 2.8 mg DNase I protein (as active ingredient) per dose, 2.3- 2.7 mg DNase I protein (as active ingredient) per dose or 2.4- 2.6 mg DNase I protein (as active ingredient) per dose. In some embodiments the effective dosage of prh DNase I is administered once daily, once every 2 days, once every 2-5 days, once every 2-10 days or more. In some embodiments the effective dosage of prh DNase I is administered 2, 3, from 2-4, from 2-6, from 2-8 or more times per day. In some embodiments of the invention, 2.5 mg of prh DNase I is administered via pulmonary administration once daily for treatment of cystic fibrosis.

[0207] Excess DNA accumulated in secretions, fluids or tissues has been associated with a number of pathological and disease-relate conditions, not only in pulmonary conditions, but also in conditions such as sepsis, infertility and metastatic spread of cancer. The prh DNase I of the present invention, administered so as to reach the site of excess extracellular DNA, can effectively lyse such extra cellular DNA and thereby reduce the severity of, alleviate the symptoms of, treat, prevent or cure the DNA- relate condition. Thus, according to some embodiments of the present invention, the disease or condition is associated with excess extra-cellular DNA in a fluid, secretion or tissue of said subject, and said administering said pharmaceutical compositions results in nucleolysis of said extra-cellular DNA.

[0208] In yet other embodiments, the plant expressed human recombinant DNase can be used for treatment or prevention of male infertility (see, for example US20110033438 to Bartoov et al. and US20070259367 to Ax et al.), and for the treatment or prevention of infectious disease caused by bacteria, virus, fungi and protozoa, treatment or prevention of sepsis (e.g. bacterial sepsis), treatment or prevention of tumors, both primary and metastatic, for prevention or reduction of metastatic growth, treatment and prevention of atherosclerosis, diabetes, delayed-type hypersensitivity reaction, treatment and prevention of diseases caused by somatic cell mutation and for enhancing longevity in an organism (see, for example US20080004561 to Genkin et al). Treatment of male infertility by phr DNase I may be directed towards reducing the amount of DNA in semen samples, as taught, for example, by Ax et al, via ex-vivo providing the semen samples with prh DNase I. In other embodiments, treatment of male infertility, tumors, metastatic transformation and growth, atherosclerosis, uterine and endometrial disorders, sepsis, viral, bacterial, fungal and protozoan infections, delayed type hypersensitivity reaction and diseases caused by somatic cell mutation is directed to reducing the amount of DNA in a subject, in-vivo, and the DNase can be administered by any route or method suitable for delivery of the DNase to the desired target within the subject's body.

[0209] Thus, in some embodiments, there is provided method of treating a subject, wherein the subject is suffering from a disease or condition selected from the group consisting of male infertility, metastatic cancer, a viral, bacterial, fungal or protozoan infection sepsis, atherosclerosis, diabetes, delayed type hypersensitivity and a uterine disorder, by administering to the subject an effective amount of the prh DNase I of the invention. Methods for such in-vivo administration include, but are not limited to: oral administration, inhalation, intraperitoneal, intravenous, subcutaneous, intramuscular injection or any other form of systemic administration (see, for example, US20110033438 or US20080004561). In some embodiments of the invention the prh DNase I is administered as a pharmaceutical composition.

[0210] The term "treating" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

[0211] As used herein, the term "preventing" refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0212] As used herein, the term "subject" includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.

[0213] As used herein the phrase "treatment regimen" refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue). The type of treatment can include a surgical intervention (e.g., removal of lesion, diseased cells, tissue, or organ), a cell replacement therapy, an administration of a therapeutic drug (e.g., receptor agonists, antagonists, hormones, chemotherapy agents) in a local or a systemic mode, an exposure to radiation therapy using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.

[0214] It is expected that during the life of a patent maturing from this application many relevant vectors, promoter elements, plant cells and carriers will be developed and the scope of the terms provided herein is intended to include all such new technologies a priori.

[0215] As used herein the term "about" refers to ± 10 %.

[0216] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

[0217] The term "consisting of' means "including and limited to".

[0218] The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0219] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0220] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0221] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0222] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0223] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0224] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0225] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES



[0226] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0227] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1, 2, 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996) Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

EXAMPLE I: CONSTRUCTION OF THE PLANT DEOXYRIBONUCLEASE I EXPRESSION CONSTRUCT



[0228] cDNA encoding the human deoxyribonuclease I (DNase I) protein (EC 3.1.21.1 GenBank: NM_005223) was optimized and synthesized by GENEART AG (Regensburg, Germany). The codon usage without the 22 amino acid leader peptide (e.g. endoplasmic reticulum target signal peptide) was adapted to the codon bias of Nicotiana tabacum genes. During the optimization process the following cis-acting sequence motifs were avoided: Internal TATA-boxes, chi-sites and ribosomal entry sites, AT-rich or GC-rich sequence stretches, RNA instability elements ("killer motifs"), Repeat sequences and RNA secondary structures, splice donor (cryptic) and acceptor sites, branch points. In addition, regions of very high (>80%) or very low (<30%) GC content were avoided.

[0229] The nucleotide sequence of the native human DNase I leader peptide (endoplasmic reticulum target signal peptide) (Figure 1, first 22 amino acids) of the full length human DNase I protein (GenBank: NM_005223) was replaced with a nucleotide sequence encoding the 33 amino acid endoplasmic reticulum targeting signal peptide (leader peptide) of the Arabidopsis ABPI protein (marked in Figure 1, SEQ ID NO: 4). This ER targeting signal peptide provides efficient targeting of DNase I to the secretory pathway and is then cleaved from the polypeptide, by signal peptidase, once the protein has been translocated into the endoplasmic reticulum. In some cases the cleavage of ABPI signal is not complete and the last (C-terminal) amino acid Glycine of the signal peptide remains, being added to the N terminal of the mature DNase I protein. As the optimized DNase I coding sequence does not contain any compartment (organelle) localization signal at the 3' terminus, the recombinant DNase I, absent a C-terminal localization signal is directed to the plant cell apoplast (the extracellular space surrounding the plant cell symplast), and when expressed in plant cell cultures, is subsequently secreted into the culture medium. SEQ ID NO: 1 represents the complete coding sequence of the expressed recombinant human DNase I polypeptide, including the N-terminal ABPI endoplasmic reticulum targeting signal peptide (SEQ ID NO: 4) and the human DNase I protein (SEQ ID NO: 6).

EXAMPLE 2: EXPRESSION OF RECOMBINANT hDNase I IN PLANTS


Transient expression system in N.benthamiana



[0230] The use of plant viral vectors was chosen in this case as an alternative to transgenic plants, allowing for the rapid, high level transient expression of proteins in mature whole plants.

[0231] The protein of interest was expressed from a strong subgenomic viral coat protein promoter. The system relies on transient amplification (by agroinfection) of viral vectors delivered to a plant by Agrobacterium, in which the plant functional promoter and the cDNA encoding a viral replicon are transferred as T-DNA from Agrobacterium into plant cells. The T-DNA is transcribed in-planta by the plant promoter to generate biologically active viral RNA that initiates self replication.

[0232] For the transient expression a 3-vector recombination system based on the system previously developed as described (Gleba et al., Vaccine 23 2042-2048, 2005) was employed. DNase I cDNA was inserted into one of the vectors, and the two other vectors contained genes for construction of the whole viral replicon (RdRp and Integrase), thus generating a biologically active viral RNA capable of initiating self replication.

Transfection of whole plants-



[0233] N. Benthamiana plants were germinated and grown in commercial mix soil (Givaat Ada, IL) supplemented with granular slow release fertilizer (Scott Marysville, OH) under a long day (16h light / 8h dark) light regime at 24°C-25°C.

[0234] Agrobacteria were transformed with the pICH20866-DNaseI based replicon vector system using electroporation (2500V, 5msec) [den Dulk-Ra, A. and Hooykaas, P.J. (1995) Methods Mol. Biol. 55:63-72]. Plants were infiltrated with Agrobacteria containing the 3 ICON plasmids by vacuum infiltration with standard methods known in the art. Briefly, N. benthamiana plants 5-6 week old were infiltrated by immersing all aerial plant organs into a bacterial suspension and were placed in a vacuum chamber. A minus (-) 0.8 bar vacuum was applied for 1 minute, followed by a quick return to atmospheric pressure. Plants were returned to the greenhouse for additional 5-7 days under the same growth conditions.

[0235] Screening for targeting to plant cell compartments was accomplished using the 3 vector recombination system described above, with the DNase I-based vector including a sequence encoding an N-terminal Arabidopsis ABPI signal peptide (marked in Figure 1, SEQ ID NO: 4), followed by the nucleotide sequence of human DNase I, and further followed by one of the below:
  1. 1) A nucleotide sequence (SEQ ID NO: 14) encoding the endoplasmic reticulum retention signal SEKDEL (SEQ ID 13), allowing retrieval of the expressed protein from the Golgi apparatus, effectively maintaining the protein in the endoplasmic reticulum;
  2. 2) A nucleotide sequence (SEQ ID NO: 16) encoding for the vacuole targeting signal peptide GLLVDTM (SEQ ID 15), allowing transfer of the expressed protein from the Golgi apparatus to the cell vacuole; or
  3. 3) No addition of any compartment localization signal at the 3' terminus of the nucleic acid sequence, directing localization of the expressed DNase I protein to the plant cell apoplast, by default.

Tobacco Plant Extract:



[0236] Samples of Nicotiana benthamiana leaves were harvested 5 days post infiltration and extracted in Laemmli buffer for SDS-PAGE, or in extraction buffer (25mM HEPES-NaOH, 40mM CaCl2, 40mM MgCl2, pH 7.5) for assays of catalytic activity and ELISA of the plant expressed protein.

Tobacco Plant Extract Purification:



[0237] Human DNase I protein from plant extracts was purified by centrifugation of the homogenized plant mass, discarding the plant debris. The supernatant was heated to 60°C for 10 minutes and filtered through a 3 µm filter, followed by filtration through a 0.65 µm filter. The filtrate was further purified on a hydrophobic interaction chromatography resin (Butyl 650C, Toyopearl) followed by further purification on an anion exchange chromatography resin (Poros 50HQ). The final material was filtered for sterility (0.2 µm).

Stable expression in N. tabacum BY2 cells



[0238] Agrobacterium mediated transformation is widely used to introduce foreign genes into a plant cell genome. This technique is based on the natural capability of the agrobacterium to transform plant cells by transferring a plasmid DNA segment, the transferred DNA (T-DNA), into the host cell genome. Using this approach, a T-DNA molecule, consisting of a foreign gene and its regulatory elements, is randomly introduced into the plant genome. The site of integration, as well as the copy number of the gene insertions is not controlled, thus the transformation process results in a 'pool' of transgenic cells composed of cells with various levels of expression of the transgene. The transgenic 'pool' is subsequently used for clone isolation. Clone isolation results in the establishment of many single cell lines, from which the clone with the highest expression level of the foreign gene is then selected.

[0239] BY2 (Bright Yellow 2) suspension culture was co-cultivated, for 48 hours, with the Agrobacterium tumefactiens EHA105 strain carrying the vector harboring the DNase I gene and the neomycin phosphotransferase (NPTII) selection gene.

[0240] Subsequently, the cells were kept in media supplemented with 50mg/L of Kanamaycin and 250mg/L Cefotaxime. The NPTII gene confers resistance to Kanamycin, thus only NPTII positive BY2 cells survive in this selection media. The Cefotaxime was used to selectively kill the agrobacterium, the plant cells being resistant to this antibiotic. Once a nicely growing transgenic cell suspension was established, it was used for screening and isolating individual cell lines. To allow for the selection of individual cell lines, aliquots of highly diluted cell suspension were spread on solid BY-2 medium. The cells were then grown until small calli developed. Each callus was then re-suspended in liquid culture. Media was then sampled and evaluated for DNase I levels. The lines that secreted relatively high DNase I levels were then further re-analyzed and compared for DNase I levels ending with the final selection of candidate DNase I expressing lines.

[0241] Media samples of transformed BY2 cells expressing the human DNase I protein were collected and when required, concentrated 5x by centrifugal filters (Amicon Ultra, 10K, #UFC501096). DNase I catalytic activity in cell's media was determined by DNA-Methyl Green assay and compared to total DNase I amount, determined by Enzyme-linked immunosorbent assay (ELISA 1) as described hereinbelow.

Protein Purification from BY-2 cells:



[0242] Recombinant human DNase-I protein secreted from tobacco suspension plant cells was purified by the following steps: at the end of the fermentation the intact tobacco cells were separated from the media by filtration using 100 mesh filter bags. The cells were discarded and the media containing the prhDNAse-I was collected for additional filtration with 0.2µm filter sheets using filter-press apparatus. The DNase in the filtrated media was further purified by two steps of chromatography columns of an anion exchange resin (Poros 50HQ, Applied Biosystems, USA) followed by hydrophobic interaction chromatography of Phenyl 650C resin (Toyopearl, Japan). The purified DNase collected from the last column was 0.2 µm filtrated and stored at 4° C.

Gel electrophoresis:



[0243] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) separates proteins primarily by their molecular weight, proteins migrating as a linear function of the logarithm of their molecular weight in SDS-PAGE. For estimating the molecular weight of prh DNase I, migration on SDS-PAGE was compared to commercial molecular weight standard proteins (New England BioLabs; cat No. P7708S) and to the migration of commercially available, mammalian-cell derived recombinant human DNase I, Pulmozyme® (Genentech, CA), expressed in CHO cells. prh DNase I and Pulmozyme were analyzed by 15% Tris-Glycine SDS-PAGE. Electrophoresis was performed using Criterion™ cell vertical electrophoresis apparatus (Bio-Rad Lab.) with premixed electrophoresis Tris-Glycine-SDS running buffer (Bio-Rad Laboratories). Fifteen percent acrylamide gels were prepared using premixed solutions of 40% Acrylamide/Bis and 10% SDS solution. Identification of the proteins was performed using two methods of detection: Coomassie brilliant blue staining and Western blot analysis using specific antibodies and Enhanced Chemical Luminescence (ECL).

Coomassie Blue Staining



[0244] Following SDS-PAGE, gels were stained with Bio-Safe™ Coomasie Stain (Bio-Rad) according to manufacturer's instructions.

Western Blotting


Western blot



[0245] Antibodies: Detection of rh DNase1 was carried out by using either
  1. (a) whole antiserum obtained from rabbits immunized against Pulmozyme®. These antibodies were prepared according to a standard polyclonal antibody preparation protocol including four immunizations with 3 mg antigen per rabbit, and collection of the serum after the fourth immunization; or
  2. (b) affinity purified rabbit anti- prh DNase I and goat- anti- prh DNase I acquired following immunization of rabbits and goats with prh DNase and affinity purification on a Pulmozyme® column. Thus, the purified antibodies detect the common backbone sequence of Pulmozyme and prhDNase. These antibodies were prepared by GenScript USA Inc in accordance to GenScript standard polyclonal antibody preparation protocol including four immunizations with 3mg antigen per rabbit and 8 mg antigen per goat, and collection of the serum after the forth immunization.


[0246] PAGE and Blotting: Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane using iBlot™ Dry Blotting System (Invitrogen). Transfer was performed at room temperature for 8 min. Then, the membrane was blocked with 5%(v/w) non-fat dry milk powder in PBS supplemented with 0.05% (v/v) Tween-20, washed with PBS supplemented with 0.05% (v/v) Tween-20, bound to the primary antibody, followed by wash with PBS supplemented with 0.05% (v/v) Tween-20 and incubation with secondary HRP-conjugated antibody. Primary antibody was diluted 1:5000 (whole antiserum) or 1:10000 (affinity purified rabbit and goat anti prh DNase I) in 1% (w/v) non-fat dry milk powder in PBS supplemented with 0.05% (v/v) Tween-20, and the secondary antibody-goat anti-rabbit or rabbit anti-goat HRP conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., PA; cat No. 111-035-003 and cat No. 305-035-003respectively) was diluted 1:10,000 in 1% (w/v) non-fat dry milk powder in PBS supplemented with 0.05% (v/v) Tween-20. Enhanced Chemi-Luminescence (ECL) reaction was performed with ECL detection kit (Pierce; cat. No. 34080). The immunoreactive bands were detected and documented using the Molecular Imager Gel Doc XR System (Bio-Rad Laboratories, UK) for time intervals up to 60 seconds, as needed.

Isoelectric focusing



[0247] Isoelectric Focusing (IEF) separates proteins on the basis of their charge in an electrical field. The protein migrates in a pH gradient generated by an electric field until it reaches a point in which its net charge equals zero, and that pH reflects its isoelectric point (pI). To identify the pI of rh DNase I, protein was analyzed by polyacrylamide IEF gels using an XCell SureLock Electrophoresis Cell equipped with a Powerpac power supply (BIO-RAD) and by Imaged capillary isoelectric focusing (iCE) using an iCE280 analyzer (Convergent Bioscience, Toronto Canada). Pre-cast polyacrylamide IEF gels with a pH range of 3-7 were obtained from Invitrogen (Novex®, Invitrogen, cat No. EC6645BOX/EC66452BOX); buffers were obtained from Invitrogen (anode buffer: Novex®, cat. No LC5300; cathode buffer: Novex®, cat. No. 5370; sample buffer: Novex®, cat. No. 5371); pI protein standards were obtained from SERVA (cat. No. 39212). Electrophoresis conditions: 100mV-1h, 200mV-1h, 500mV-1.5h. Bands were visualized by Bio-Safe™ Coomassie Stain (Bio-Rad, cat No. 161-0787) according to the manufacturer's instructions. The pI profile of rh DNase I was estimated using the pI protein standards, and then the banding profile was compared to that of Pulmozyme®.

[0248] For imaged capillary isoelectric focusing analysis, a sample contained prh DNase I, pI markers 3.59 and 5.85 (protein simple, cat No. 102222 and 102225, respectively), 0.35% methylcellulose (protein simple, cat No. 101876), and SERVALYT pH 3-7 (SERVA, cat No. 42945.01) was introduced to the capillary through an autosampler (PrinCE) and was focused in a capillary column under high voltage. The focusing process was monitored in real-time using whole column imaging detection (WCID). The resolved charge species appear as electrophoretic peaks that are detected at a fixed wavelength of UV280 nm. pI values and peak areas for each species were determined from the resultant electropherograms.

Mass Spectrometry


Molecular Weight Analysis



[0249] Molecular weight analysis was accomplished by mass spectrometry (MS) of rh DNase I, using a matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometer and sinapinic acid as a matrix. The equipment was calibrated using molecular weight standards. About 2.5 micrograms of rh DNase were used for mass analysis.

Amino Acid Sequence and structural characterization



[0250] prh DNase I sequence was analysed using reverse-phase (RP-) HPLC coupled to mass spectrometry.

[0251] The protein samples were reduced using 2.8 mM DTT (60°C for 30 minutes), modified using 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark, room temperature for 30 minutes) and digested in 10% ACN and 10 mM ammonium bicarbonate with modified trypsin (Promega) or with chymotrypsin overnight at 37°C in a 1:50 enzyme-to-substrate ratio. 3% of the resulting peptides were resolved by reversed-phase liquid chromatography on a 0.075X200-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr Maisch, GmbH, Germany). Peptides were eluted with a linear 60 minutes gradient of 5 to 45% followed by 15 minutes at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25 µL/min. On-line mass spectrometry was performed by an ion-trap mass spectrometer (Orbitrap, Thermo) in a positive mode using repetitively full MS scan followed by collision-induced dissociation (CID) of the 7 most dominant ions selected from the first MS scan. The mass spectrometry data was analyzed using the Sequest 3.31 software (J. Eng and J.Yates, University of Washington and Finnigan, San Jose), compared to the human reference sequence.

[0252] Further sequencing and structural characterization data was obtained from trypsin digestion of prhDNase I, with or without a further digestion with PNGaseA in the presence and absence of DTTand analysis by mass spectrometry

Disulfide Bonds and Free Sulfhydryl Content



[0253] Human DNase-1 sequence includes four cysteine residues and contains two disulfides bonds. It was expected that the structure a recombinant human DNase-1 expressed in plant cells (prhDNase I), will be similar to the authentic human protein.

[0254] The assessment of the prhDNase I thiol content was obtained by using the Ellman's method, which is a rapid and sensitive method for quantitative analysis of the total free thiols in peptides and proteins. The number of free thiols found on prhDNase I also indicates the number of cysteine residues that are engaged in disulfide bonds.

[0255] Free thiols analysis of prhDNase I and Pulmozyme® -was carried out according to standard Ellman's method under denaturing conditions. The reaction was performed by mixing the protein solution in sodium phosphate buffer with 6M Guanidine Hydrochloride and the active substrate DTNB [5, 5'-Dithiobis (2-nitrobenzoic acid)]. The solutions were incubated at ambient conditions for 5-15 min. During the reaction, TNB (2-nitro-5-thiobenzoic acid) was released. A reaction of 1 mole DTNB with 1 mole thiol releases 1 mole of TNB anion with an extinction coefficient of 13,700 M-1 cm-1 at 412nm. The number of thiols was calculated as direct ratio between TNB concentration and prhDNase I concentration in the analyzed sample. A protein, containing free thiols was used as positive control and DNase I formulation buffer (1mM CaCl2, 150mM NaCl, pH 6.1-6.5) was used as blank for analysis.

Glycan structure of rh DNase


Glycan analysis of the plant-expressed human DNAseI.



[0256] Samples of recombinant human DNAseI protein product from the transformed cells were reduced with DTT alkylated with iodoacetamide, and separated on a 12.5% SDS-PAGE. Bands corresponding to the correct molecular weight were excised and subjected to glycan analysis consisting of trypsin digestion followed by peptide extraction and by both PNGase A and PNGase F digestion. PNGase A digestion releases all the N-linked glycans and PNGase F release all glycans except those containing α 1-3 core fucose.

[0257] The free glycans were released, purified and then labeled with the fluorescent reagent anthranilamide (2AB) followed by removal of excess 2AB.

[0258] Glycans were separated using a TSK gel Amide 80 normal phase HPLC and detected using a fluorescence detector. A Dextran hydrolysate served as a ladder for calculation of glucose unit (GU) values. Glycan profile is constructed by calculating the relative peak areas from the chromatogram of the PNGase A digestion. Assignment of the glycans is established by calculation of the GU values of the peaks found in both endoglycoside digestions and based on additional various exoglycoside digestions, comparison to known databases and confirmation of results by mass spectrometric methods.

Reverse Phase-High Performance Liquid Chromatography (RP-HPLC)



[0259] RP-HPLC was used as an analysis method for the determination of purity of hr DNase I. The analysis employed a Jupiter 3µ, C18, 300Å, 250x4.6mm column by Phenomenex. Analyses were performed on a Dionex, Ultimate 3000 HPLC system equipped with a photodiode array detector. Chromatograms were routinely recorded at 2 wavelengths, 214 and 280nm.

Elution solvents:



[0260] 

Solvent A: 1 Liter, 0.1% TFA/H2O (HPLC grade, J.T.Baker, cat # 4218-03). Solvent A was prepared by adding 1.0 mL of TFA (Sigma, cat. # T6508) to 0.999 liter of water (HPLC grade, J.T. Baker, cat. # 4218-0.3).

Solvent B: 1 Liter, 0.1% TFA/Acetonitrile (HPLC grade, Sigma, cat. # 34888). Solvent B was prepared by adding 1.0 mL TFA (Sigma, cat. # T6508) to 0.999 liter of acetonitrile (HPLC grade, Sigma, cat. # 34888).


Chromatographic method:



[0261] Chromatographic separations were performed on a Jupiter 3µ, C18, 300Å, 250x4.6mm column (Phenomenex, cat. 00G-4263-E0). Chromatograms were obtained using a linear gradient of 5%B for 3min, followed by 5%B to 43%B for 10min, followed by 43%B to 55%B for 30min, followed by 55%B to 95%B for 10min followed by 95%B for 3min followed by 95%B to 5%B for 2min, followed by 5%B for 21 min at a flow rate of 1 ml/min and at a temperature of 55°C. Each analysis sequence was commenced with a run of DNase I formulation buffer (1mM CaCl2 · 150mM NaCl, pH 6.1-6.5) as a blank for rh DNase I.

[0262] Samples of prh DNase I (25µg) in DNase I formulation buffer were analyzed. 25µg of the rh DNase I was pipetted into a glass insert (250 µL), which was inserted into an HPLC auto-sampler glass vial.

Results


Characterization ofplant-expressed hr-DNase I:



[0263] Yields of hr DNase I expressed in plant cells of whole tobacco plants varied with targeting to different plant organelles. Figure 2 shows the differences in yield of plant expressed human DNase I (relative to that of apoplast targeting) when phr DNase I was expressed from a DNase I construct encoding an N-terminal ER targeting signal peptide only (APO) directing the protein to the apoplast, from a DNase I construct encoding an N-terminal ER targeting signal peptide and a C-terminal vacuolar targeting signal peptide (VAC), or from a DNase I construct encoding an N-terminal ER targeting signal peptide and a C-terminal ER retention signal peptide, targeting the protein for retention in the ER (ER). Clearly, targeting to the apoplast results in the highest yield of these three expression options. Further, Figure 2 also reveals the consistent correlation between levels of immunoreactive DNase I protein and catalytic activity.

[0264] Figure 3 shows an exemplary result of the migration of prh DNase I on SDS-PAGE (lanes 6-9), compared with mammalian cell expressed recombinant human DNase I (Pulmozyme®) (lanes 1-4). The plant expressed prh DNase I consists of one major band at a molecular weight of ∼30 KDa, migrating similarly that of the commercial preparation. The prh DNase I preparation did not contain any detectable contamination, indicating a high purity. An apparent slight difference in migration characteristics was observed between the prh DNase I and the commercial preparation, with the plant expressed DNase I appearing to migrate faster. Without wishing to adhere to a single hypothesis, one possible explanation for such a difference in migration characteristics could be a difference in glycosylation patterns between the plant and mammalian cell-expressed enzymes. In order to compared for consistency between expression of phrDNase I from plants and plant cell culture, 3 batches of prh DNase I extracted from whole plants and 3 batches of prh DNase I extracted from cultured cells were analyzed on SDS-PAGE, and were observed to have the same, characteristic pattern of migration on the gel.

[0265] Figure 4 shows an exemplary result of immunological cross reactivity of the prh DNase I separated on SDS-PAGE (lanes 6-9), compared with the commercial, mammalian cell expressed recombinant human DNase I (Pulmozyme) (lanes 1-4), when reacted with unfractionated rabbit anti-human DNase I immune serum. The cross-reactive plant-expressed prh DNase I protein migrated as the 30 kDa molecular weight, similar to the commercial preparation. Further, note that the plant-expressed prh DNase I preparation does not contain any other detectable DNase I-related immunoreactive species, indicating high purity of the preparation. Three batches of DNase extracted from whole plant and 3 batches of DNase extracted from cultured cells were analyzed and have shown the same migrating characterstics. In addition, the same results were obtained using affinity purified rabbit anti- prh DNase

[0266] Figures 5A-5D shows a typical pI analysis of the plant-expressed prh DNase I as compared to Pulmozyme®. The rh DNase I is characterized by 3 bands; two major isoforms ranged between 4.2-4.5 and a minor isoform with a slightly lower pI (Fig. 5A, lanes 2 and 3 and 5B, lane 2). This is in contrast to the commercial standard, Pulmozyme® (Fig. 5A, lane 1 and 5B, lane 3), which is characterized by a variety of isoforms ranging from pI 3.5 to pI 4.5. Without wishing to adhere to a single hypothesis, one possible explanation for such a difference in pI could be a difference in glycosylation patterns between the plant and mammalian cell-expressed enzymes. Three batches of DNase extracted from whole plant and 3 batches of DNase extracted from cultured cells from cultures of different transformed BY2 lines were analyzed, and demonstrated the same migration pattern upon pI analysis. pI values for each species were determined from imaged capillary isoelectric focusing electropherograms (Figs. 5C and 5D). A main peak (MP, pI 4.41) and two acidic peaks (pre-MP1 and pre-MP2 with pI of 4.27 and 4.21) were observed, consistent with the pI profile observed in the IEF gels (Figs. 5A and 5B).

[0267] Figures 6A and 6B depict an analysis of the molecular weight of plant-expressed prh DNase I by MALDI-ToF, showing the entire spectrum from 20000 to 180000 m/z (6A) or an enlargement of the prh DNase I peak at about 32000 m/z (6B). The molecular weight of the prh DNase I enzyme extracted from the plant cells was approximately 32,070 Da, while the theoretical molecular weight of the prh DNase I polypeptide, based on the proteins' 261 amino acid sequence, is 29,311 Da. Without wishing to adhere to a single hypothesis, one likely explanation for such a difference in the chromatographic characteristics is the difference in glycosylation patterns. Therefore, glycan structures may add the remaining ∼2760 Daltons. Three batches of prh DNase I extracted from cultured cells were analyzed and have shown similar characteristics.

[0268] The difference in the molecular weight of the three phr DNase I main isoforms seen in the MALDI analysis was approximately 200 Da. (Fig 6B) This may correspond to a difference in glycan structures that can be attributed to one or two residues of N-Acetylglucosamine (203 Da). The same indication can be found in the variability of glycan structures.

[0269] Thus, the results of the mass spectrometry analysis confirm the molecular weight determination from the SDS-PAGE analysis (see Figure 3), and both are consistent with a molecular weight of the prh DNase I comparable to the calculated molecular weight of the expressed polypeptide, and consistent with the molecular weight of mammalian cell expressed recombinant human DNase I (Pulmozyme®).

Amino acid sequence of plant-expressed rh DNase I



[0270] Mass spectrometry analysis of the peptides generated by trypsin and chymotrypsin digestion of plant-expressed rh DNase I showed over 97.3% coverage of the 261 DNase I amino acid sequences that matched the predicted amino acid sequence based on the DNA sequence of the expression cassette (SEQ ID NO: 6). Figure 7 represents the composite amino acid sequence derived from the overlapping peptides, with unconfirmed amino acids, putative glycosylation sites bolded and underlined, and the additional N-terminal glycine marked. When compared to the peptide sequence of the native human enzyme (SEQ ID NO: 6, GenBank Accession No: NP_005214.2) it is apparent that the amino acid sequence of plant-expressed rh DNase I is identical to that of commercial, mammalian cell expressed dornase alfa (Pulmozyme®), and in some embodiments, with an additional glycine residue at position 1 at the N terminus, derived from the ABPI signal peptide. Indeed, further HPLC and MS analysis of a trypsin digest and chymotrypsin sub-digest of the plant-expressed rh DNase I provided a full 261 amino acid sequence, confirming the accuracy of the sequence derived from overlapping peptides (FIG 7) and the identity of the amino acid sequence of the plant expressed rh DNase I and that of commercial, mammalian cell expressed dornase alfa (Pulmozyme®), with the addition of a glycine residue at position 1 at the N terminus, derived from the ABPI signal peptide. Furthermore, the analysis of the trypsin digest of prhDNAse I treated and not treated with PNGase A revealed the presence of glycan sites on Asp 19 and Asp 107 as supported by the glycan analysis data in Fig 8 (below). In addition, disulphide bridges were observed between Cys102 and Cys105 and between Cys174 and Cys210.

[0271] The overlapping peptides are presented in the following coverage table (Table V), in which the first amino acid of plant-expressed rh DNase I, Gly, is numbered as amino acid 33, and where amino acids 1-32 are comprised of the ABPI signal sequence (SEQ ID NO: 4), which are cleaved from the expressed polypeptide following transfer to the endoplasmic reticulum. Table Va shows peptides produced by trypsin and PNGase digestion. In this table Gly, is denoted as amino acid number 1
Table V- Peptides Identified Following Digestion with Trypsin and Chymotrypsin
Position% MassSEQ ID NO:Sequence
33 - 48 5.325060547 17 GLKIAAFNIQTFGETK
34 - 39 2.029654905 18 LKIAAF
34 - 44 3.878161246 19 LKIAAFNIQTF
34 - 48 5.150347722 20 LKIAAFNIQTFGETK
35 - 48 4.803860015 21 KIAAFNIQTFGETK
36 - 42 2.378969069 22 IAAFNIQ
36 - 44 3.139192685 23 IAAFNIQTF
36 - 45 3.313905509 24 IAAFNIQTFG
36 - 48 4.411379161 25 IAAFNIQTFGETK
38 - 48 3.847234909 26 AFNIQTFGETK
39 - 48 3.629578364 27 FNIQTFGETK
40 - 44 1.906776692 28 NIQTF
40 - 48 3.178963169 29 NIQTFGETK
42 - 48 2.483049813 30 QTFGETK
43 - 48 2.090680443 31 TFGETK
49 - 57 3.019451292 32 MSNATLVSY
49 - 64 5.499783699 33 MSNATLVSYIVQILSR
53 - 64 4.264530922 34 TLVSYIVQILSR
55 - 64 3.608434794 35 VSYIVQILSR
58 - 64 2.538602758 36 IVQILSR
58 - 65 3.038226108 37 IVQILSRY
59 - 64 2.192115051 38 VQILSR
60 - 64 1.888571065 39 QILSR
63 - 74 4.439055196 40 SRYDIALVQEVR
65 - 70 2.124510303 41 YDIALV
65 - 71 2.516879673 42 YDIALVQ
65 - 73 3.215808036 43 YDIALVQEV
65 - 74 3.694099266 44 YDIALVQEVR
65 - 75 4.046539923 45 YDIALVQEVRD
65 - 78 5.079638575 46 YDIALVQEVRDSHL
65 - 83 6.477641206 47 YDIALVQEVRDSHLTAVGK
66 - 71 2.017256323 48 DIALVQ
66 - 74 3.194475917 49 DIALVQEVR
66 - 83 5.978017857 50 DIALVQEVRDSHLTAVGK
67 - 74 2.84203526 51 IALVQEVR
68 - 73 2.017256323 52 ALVQEV
68 - 74 2.495547553 53 ALVQEVR
69 - 74 2.277891008 54 LVQEVR
70 - 74 1.931403301 55 VQEVR
72 - 83 4.019031885 56 EVRDSHLTAVGK
74 - 83 3.320103521 57 RDSHLTAVGK
75 - 82 2.449331437 58 DSHLTAVG
75 - 83 2.841812291 59 DSHLTAVGK
75 - 106 10.83964091 60 DSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGR
76 - 83 2.489371635 61 SHLTAVGK
83 - 96 4.963013347 62 KLLDNLNQDAPDTY
84 - 89 2.149025427 63 LLDNLN
84 - 90 2.541394797 64 LLDNLNQ
84 - 91 2.893835453 65 LLDNLNQD
84 - 92 3.111491998 66 LLDNLNQDA
84 - 95 4.070909143 67 LLDNLNQDAPDT
84 - 96 4.570532493 68 LLDNLNQDAPDTY
84 - 97 4.990478738 69 LLDNLNQDAPDTYH
84 - 98 5.490102088 70 LLDNLNQDAPDTYHY
84 - 106 8.056098967 71 LLDNLNQDAPDTYHYVVSEPLGR
84 - 110 9.564293519 72 LLDNLNQDAPDTYHYVVSEPLGRNSYK
84 - 112 10.43796913 73 LLDNLNQDAPDTYHYVVSEPLGRNSYKER
85 - 99 5.447158367 74 LDNLNQDAPDTYHYV
86 - 96 3.877557079 75 DNLNQDAPDTY
86 - 106 7.363123553 76 DNLNQDAPDTYHYVVSEPLGR
87 - 106 7.010682896 77 NLNQDAPDTYHYVVSEPLGR
88 - 106 6.661257247 78 LNQDAPDTYHYVVSEPLGR
90 - 106 5.965343891 79 QDAPDTYHYVVSEPLGR
91 - 106 5.572974522 80 DAPDTYHYVVSEPLGR
92 - 98 2.654536986 81 APDTYHY
92 - 106 5.220533865 82 APDTYHYVVSEPLGR
93 - 106 5.00287732 83 PDTYHYVVSEPLGR
94 - 106 4.705509252 84 DTYHYVVSEPLGR
95 - 106 4.353068596 85 TYHYVVSEPLGR
97 - 102 2.246976996 86 HYVVSE
97 - 104 2.890832771 87 HYVVSEPL
97 - 105 3.065545596 88 HYVVSEPLG
97 - 106 3.543836826 89 HYVVSEPLGR
97 - 107 3.893262475 90 HYVVSEPLGRN
97 - 110 5.052031378 91 HYVVSEPLGRNSYK
98 - 106 3.12389058 92 YVVSEPLGR
99 - 104 1.971263176 93 VVSEPL
99 - 107 2.97369288 94 VVSEPLGRN
99 - 110 4.132461783 95 VVSEPLGRNSYK
111 - 115 2.22867221 96 ERYLF
113 - 120 3.286263893 97 YLFVYRPD
113 - 122 3.982177249 98 YLFVYRPDQV
113 - 123 4.248841949 99 YLFVYRPDQVS
113 - 124 4.466498494 10 YLFVYRPDQVSA
113 - 126 5.122483137 101 YLFVYRPDQVSAVD
113 - 128 5.888771186 102 YLFVYRPDQVSAVDSY
113 - 129 6.388394536 103 YLFVYRPDQVSAVDSYY
113 - 131 7.240458542 104 YLFVYRPDQVSAVDSYYYD
113 - 132 7.592899198 105 YLFVYRPDQVSAVDSYYYDD
113 - 133 7.767612023 106 YLFVYRPDQVSAVDSYYYDDG
114 - 128 5.389147837 107 LFVYRPDQVSAVDSY
114 - 129 5.888771186 108 LFVYRPDQVSAVDSYY
115 - 128 5.04266013 109 FVYRPDQVSAVDSY
115 - 129 5.542283479 110 FVYRPDQVSAVDSYY
116 - 123 2.952115698 111 VYRPDQVS
116 - 125 3.473316229 112 VYRPDQVSAV
116 - 126 3.825756886 113 VYRPDQVSAVD
116 - 128 4.592044935 114 VYRPDQVSAVDSY
116 - 129 5.091668284 115 VYRPDQVSAVDSYY
116 - 130 5.591291634 116 VYRPDQVSAVDSYYY
116 - 131 5.94373229 117 VYRPDQVSAVDSYYYD
116 - 132 6.296172947 118 VYRPDQVSAVDSYYYDD
116 - 133 6.470885771 119 VYRPDQVSAVDSYYYDDG
118 - 128 3.788877599 120 RPDQVSAVDSY
118 - 129 4.288500948 121 RPDQVSAVDSYY
118 - 130 4.788124298 122 RPDQVSAVDSYYY
118 - 132 5.493005611 123 RPDQVSAVDSYYYDD
140 - 150 4.037383416 124 DTFNREPAIVR
141 - 150 3.684942759 125 TFNREPAIVR
143 - 150 2.924719144 126 NREPAIVR
143 - 151 3.375334339 127 NREPAIVRF
144 - 150 2.575293495 128 REPAIVR
153 - 159 2.74066949 129 SRFTEVR
155 - 161 2.841713132 130 FTEVREF
156 - 161 2.391097937 131 TEVREF
157 - 161 2.081489517 132 EVREF
160 - 166 2.415813937 133 EFAIVPL
160 - 172 4.095594822 134 EFAIVPLHAAPGD
160 - 175 4.834451899 135 EFAIVPLHAAPGDAVA
160 - 178 5.928764639 136 EFAIVPLHAAPGDAVAEID
160 - 180 6.492908892 137 EFAIVPLHAAPGDAVAEIDAL
160 - 181 6.992532241 138 EFAIVPLHAAPGDAVAEIDALY
160 - 182 7.344972898 139 EFAIVPLHAAPGDAVAEIDALYD
160 - 183 7.648516884 140 EFAIVPLHAAPGDAVAEIDALYDV
160 - 184 8.148140234 141 EFAIVPLHAAPGDAVAEIDALYDVY
160 - 190 10.33084718 142 EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK
162 - 178 5.082765067 143 AIVPLHAAPGDAVAEID
162 - 180 5.64690932 144 AIVPLHAAPGDAVAEIDAL
162 - 181 6.146532669 145 AIVPLHAAPGDAVAEIDALY
162 - 184 7.302140662 146 AIVPLHAAPGDAVAEIDALYDVY
163 - 181 5.928876124 147 IVPLHAAPGDAVAEIDALY
163 - 190 9.267191067 148 IVPLHAAPGDAVAEIDALYDVYLDVQEK
166 - 181 4.981476363 149 LHAAPGDAVAEIDALY
167 - 180 4.135365306 150 HAAPGDAVAEIDAL
167 - 181 4.634988656 151 HAAPGDAVAEIDALY
167 - 182 4.987429312 152 HAAPGDAVAEIDALYD
167 - 183 5.290973298 153 HAAPGDAVAEIDALYDV
167 - 184 5.790596648 154 HAAPGDAVAEIDALYDVY
167 - 190 7.973303599 155 HAAPGDAVAEIDALYDVYLDVQEK
173 - 181 2.955207771 156 AVAEIDALY
173 - 190 6.293522714 157 AVAEIDALYDVYLDVQEK
179 - 190 4.460352897 158 ALYDVYLDVQEK
180 - 190 4.242696351 159 LYDVYLDVQEK
181 - 190 3.896208644 160 YDVYLDVQEK
182 - 190 3.396585295 161 DVYLDVQEK
182 - 191 3.966729241 162 DVYLDVQEKW
184 - 190 2.740600652 163 YLDVQEK
185 - 190 2.240977303 164 LDVQEK
185 - 191 2.811121249 165 LDVQEKW
185 - 199 5.533190275 166 LDVQEKWGLEDVMLM
186 - 190 1.894489595 167 DVQEK
186 - 191 2.464633542 168 DVQEKW
191 - 195 1.897439863 169 WGLED
191 - 197 2.602489733 170 WGLEDVM
191 - 198 2.94897744 171 WGLEDVML
191 - 199 3.350483324 172 WGLEDVMLM
191 - 200 3.525196149 173 WGLEDVMLMG
191 - 208 6.151953511 174 WGLEDVMLMGDFNAGCSY
191 - 210 6.933788727 175 WGLEDVMLMGDFNAGCSYVR
191 - 218 9.818443147 176 WGLEDVMLMGDFNAGCSYVRPSQWSSIR
192 - 198 2.378833494 177 GLEDVML
192 - 199 2.780339378 178 GLEDVMLM
192 - 208 5.581809564 179 GLEDVMLMGDFNAGCSY
198 - 214 5.916115429 180 LMGDFNAGCSYVRPSQW
199 - 208 3.261246422 181 MGDFNAGCSY
199 - 210 4.043081638 182 MGDFNAGCSYVR
199 - 214 5.569627722 183 MGDFNAGCSYVRPSQW
199 - 218 6.927736058 184 MGDFNAGCSYVRPSQWSSIR
200 - 208 2.859740538 185 GDFNAGCSY
200 - 210 3.641575754 186 GDFNAGCSYVR
200 - 214 5.168121838 187 GDFNAGCSYVRPSQW
200 - 215 5.434786537 188 GDFNAGCSYVRPSQWS
201 - 214 4.993409013 189 DFNAGCSYVRPSQW
203 - 214 4.190353162 190 NAGCSYVRPSQW
204 - 214 3.840927513 191 AGCSYVRPSQW
207 - 214 3.132939701 192 SYVRPSQW
207 - 218 4.491048037 193 SYVRPSQWSSIR
208 - 214 2.866275001 194 YVRPSQW
209 - 214 2.366651651 195 VRPSQW
209 - 218 3.724759988 196 VRPSQWSSIR
211 - 218 2.942924771 197 PSQWSSIR
213 - 218 2.378892004 198 QWSSIR
215 - 220 2.333010341 199 SSIRLW
219 - 224 2.158151613 200 LWTSPT
219 - 225 2.608766808 201 LWTSPTF
219 - 226 3.001136178 202 LWTSPTFQ
219 - 227 3.571280124 203 LWTSPTFQW
219 - 228 3.917767831 204 LWTSPTFQWL
219 - 231 4.914064263 205 L WTSPTFQWLIPD
219 - 234 5.750826164 206 LWTSPTFQWLIPDSAD
219 - 241 7.924230704 207 LWTSPTFQWLIPDSADTTATPTH
219 - 244 8.957129041 208 LWTSPTFQWLIPDSADTTATPTHCAY
219 - 246 9.787860928 209 LWTSPTFQWLIPDSADTTATPTHCAYDR
220 - 246 9.441373221 210 WTSPTFQWLIPDSADTTATPTHCAYDR
221 - 226 2.084504524 211 TSPTFQ
221 - 227 2.654648471 212 TSPTFQW
221 - 228 3.001136178 213 TSPTFQWL
221 - 241 7.007599051 214 TSPTFQWLIPDSADTTATPTH
221 - 244 8.040497388 215 TSPTFQWLIPDSADTTATPTHCAY
221 - 246 8.871229275 216 TSPTFQWLIPDSADTTATPTHCAYDR
225 - 246 7.687979666 217 FQWLIPDSADTTATPTHCAYDR
226 - 244 6.406632585 218 QWLIPDSADTTATPTHCAY
226 - 246 7.237364471 219 QWLIPDSADTTATPTHCAYDR
228 - 246 6.274851155 220 LIPDSADTTATPTHCAYDR
229 - 244 5.097631562 221 IPDSADTTATPTHCAY
229 - 246 5.928363448 222 IPDSADTTATPTHCAYDR
232 - 246 4.932067017 223 SADTTATPTHCAYDR
234 - 246 4.447745772 224 DTTATPTHCAYDR
235 - 244 3.264573229 225 TTATPTHCAY
236 - 246 3.785696695 226 TATPTHCAYDR
239 - 246 2.948823309 227 PTHCAYDR
245 - 250 2.060234463 228 DRIVVA
245 - 252 2.636453171 229 DRIVVAGM
245 - 253 2.982940878 230 DRIVVAGML
245 - 254 3.329428586 231 DRIVVAGMLL
245 - 255 3.807719815 232 DRIVVAGMLLR
247 - 253 2.152208992 233 IVVAGML
247 - 254 2.498696699 234 IVVAGMLL
247 - 255 2.976987929 235 IVVAGMLLR
247 - 256 3.151700754 236 IVVAGMLLRG
248 - 255 2.630500222 237 VVAGMLLR
249 - 255 2.326956236 238 VAGMLLR
250 - 255 2.023412249 239 AGMLLR
252 - 259 2.630500222 240 MLLRGAVV
254 - 266 4.11110757 241 LRGAVVPDSALPF
255 - 266 3.764619863 242 RGAVVPDSALPF
256 - 262 1.974201118 243 GAVVPDS
256 - 263 2.191857663 244 GAVVPDSA
256 - 264 2.53834537 245 GAVVPDSAL
256 - 265 2.835713438 246 GAVVPDSALP
256 - 268 4.086369477 247 GAVVPDSALPFNF
256 - 271 4.914051937 248 GAVVPDSALPFNFQAA
256 - 272 5.413675286 249 GAVVPDSALPFNFQAAY
256 - 273 5.588388111 250 GAVVPDSALPFNFQAAYG
256 - 274 5.934875818 251 GAVVPDSALPFNFQAAYGL
256 - 276 6.553981174 252 GAVVPDSALPFNFQAAYGLSD
256 - 293 12.44599823 253 GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK
267 - 272 2.185617005 254 NFQAAY
267 - 273 2.360329829 255 NFQAAYG
267 - 286 6.777625088 256 NFQAAYGLSDQLAQAISDHY
267 - 293 9.217939951 257 NFQAAYGLSDQLAQAISDHYPVEVMLK
268 - 293 8.868514302 258 FQAAYGLSDQLAQAISDHYPVEVMLK
269 - 293 8.417899107 259 QAAYGLSDQLAQAISDHYPVEVMLK
270 - 293 8.025529737 260 AAYGLSDQLAQAISDHYPVEVMLK
271 - 293 7.807873192 261 AYGLSDQLAQAISDHYPVEVMLK
273 - 280 2.547459231 262 GLSDQLAQ
273 - 282 3.111603483 263 GLSDQLAQAI
273 - 285 4.150655085 264 GLSDQLAQAISDH
273 - 289 5.646574866 265 GLSDQLAQAISDHYPVE
273 - 290 5.950118852 266 GLSDQLAQAISDHYPVEV
273 - 291 6.351624736 267 GLSDQLAQAISDHYPVEVM
273 - 292 6.698112443 268 GLSDQLAQAISDHYPVEVML
273 - 293 7.090593298 269 GLSDQLAQAISDHYPVEVMLK
275 - 293 6.569392766 270 SDQLAQAISDHYPVEVMLK
277 - 293 5.95028741 271 QLAQAISDHYPVEVMLK
279 - 293 5.211430333 272 AQAISDHYPVEVMLK
281 - 293 4.601404418 273 AISDHYPVEVMLK
283 - 293 4.037260166 274 SDHYPVEVMLK
285 - 293 3.41815481 275 HYPVEVMLK
287 - 293 2.498585215 276 PVEVMLK
Table Va- Peptides Identified Following Digestion with Trypsin and PNGase
Position SEQ ID NO:Sequence
4 - 16   278 IAAFNIQTFGETK
17 - 32   279 MSNATLVSYIVQILSR
33 - 42   280 YDIALVQEVR
43 - 51   281 DSHLTAVGK
52 - 74   282 LLDNLNQDAPDTYHYVVSEPLGR
52 - 78   283 LLDNLNQDAPDTYHYVVSEPLGRNSYK
81 - 112   284 YLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNR
81 - 118   285 YLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVR
113 - 118   286 EPAIVR
119 - 122   287 FFSR
123 - 127   288 FTEVR
128 - 158   289 EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK
215 - 223   290 IVVAGMLLR
224 - 261   291 GAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

Free Sulfhydryl Content (Ellman's Method)



[0272] Quantitative assay of the free-thiols indicated that both phr DNase I protein and Pulmozyme® lack free sulfhydryls, while the positive control detected four free sulfhydryls (thiols) as expected. These results confirm that in phr DNase I four Cysteine amino acids are bound in 2 disulfide bonds (Cys 102-Cys 105 and Cys 174-Cys210) as was suggested for DNase-1 and Pulmozyme ®.

Glycan analysis:



[0273] Preliminary analysis was performed to characterize the various glycan structures of prhDNase I produced from BY2 cell culture. Figures 8A-8C show NP-HPLC profile of glycans derived from three separate batches of prhDNase I using PNGase A to release the glycans. Release of glycans by PNGase F indicated a low abundance of glycans without core fucose structures in prhDNase I. The glycan profile of all three batches of prhDNase I indicated a similar profile. Peaks in the profiles shown in Figure 8A correlate to the detailed structures in Figure 8B indicating the relative amounts of glycans released from the batches of prhDNase I.

[0274] The glycan analysis of recombinant human DNAseI protein product from the transformed tobacco cells revealed a glycan profile comprising two main glycan peaks consisting of at least four glycan structures (e.g. peaks 3+4 and 5). Predominant glycan structures representing a high percentage of the glycan profile (over 80%) contain mannose 3-β-(1,2)xylose-α-(1,3) fucose [Fc(3)M3X] and/or mannose 4-α-(1,2)xylose (M4X) with an additional one or two N-acetylglucosamine substitution on the outer mannose sugars (Figure 8B). The other glycans contain smaller structures missing either fucose or N-acetylglucosamine substitutions (3-6%) or hybrid structures (up to 14%).

[0275] Figure 8C shows a comparison of glycans found in prhDNase I released by PNGase A compared to glycans released from Pulmozyme® by PNGase F. The wide variations in glycosylation pattern found in Pulmozyme® are due to the abundance of bi- and tri-antennary glycans with additional sialic acid residues attached, leading to a mixture of charged and uncharged glycan moieties. Clearly, phr DNase I glycosylation is more homogeneous and does not include charged glycans.

Purity ofplant-expressed rh DNase preparation:



[0276] Figures 8A- 8D represent a typical chromatogram of the plant-expressed prh DNase I, analyzed by RP-HPLC at 214nm (9A, 9B) and at 280nm (9C, 9D), after subtraction of start-of-sequence blank. Insets 9B and 9D are expanded views of the prh DNase I peak. In both chromatograms, the prh DNase I resolves as a main peak (> 93.6% purity) with a retention time of 28.3 minutes and an additional two small peaks, a pattern characterisitic of all batches of the prh DNase I tested.

[0277] Thus, the prh DNase I preparation is characterized by molecular weight, pI, free sulfhydryl content, immune cross-reactivity and amino acid sequence comparable to but distinct from that of commercially available mammalian cell expressed DNase I, and consistent with the translation product of the expressed human DNase I coding sequence of SEQ ID NO: 9. Further, the plant-expressed rh DNase I is secreted into the medium of cells in suspension culture, and can be purified to a high degree of purity, containing less than 7% impurities detected by HPLC at both 214 and 280 nm.

EXAMPLE 3: BIOLOGICAL ACTIVITY OF PLANT-EXPRESSED rh DNASE I



[0278] Biological activity of the plant-expressed rh DNase I was assayed using several assays, and compared to that of the mammalian-cell expressed commercially available human recombinant enzyme Pulmozyme® (Genentech, MA).

Methods:


DNA-Methyl Green assay



[0279] Deoxyribonuclease I (DNase I) is an endonuclease that cleaves DNA preferentially at phosphodiester linkages, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides.

[0280] Activity of DNase I was assessed by the Methyl Green enzymatic activity assay, employing DNA from Salmon Testis (Sigma cat No. D1626) complexed with the dye methyl green (Sigma cat No. M8884) as a substrate. Methyl green intercalates between the stacked bases of double-stranded DNA. Once the long DNA molecules are hydrolyzed into tetranucleotides as a result of DNase1 activity, dissociation of methyl green from the DNA occurs, the free methyl green decolorizing in a second, nonenzymatic reaction. The spontaneous decolorization of free methyl green at neutral pH is likely to result from tautomerization of the dye.

[0281] Standard curves were prepared by dilution of purified standard phrDNase I in activity Buffer (25mM HEPES-NaOH, 4mM CaCl2, 4mM MgCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5) at concentrations ranging from 0.3 to 20 ng/ml at 2-fold series dilutions. Samples and controls were prepared in a similar matter. One hundred microliters of standards, controls and samples was added in duplicates to a 96-well plate (NUNC) containing 100µl of DNA-methyl green substrate and the contents were mixed thoroughly. The plates were then sealed and agitated for 30 min at room temperature, then incubated overnight at 37°C and absorbance measured at 620nm. Absorbance was plotted versus standards concentrations and the data were fit to a 4-parameter logistic model by the nonlinear regression method of Marquardt.

[0282] DNase I activity in different expression systems was expressed as percent of the activity measured in the apoplast targeted cells (100%) (see Fig 2).

Enzyme-linked immunosorbent assay (ELISA)



[0283] Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of immunoreactive rh DNase I in a sample. Two different ELISA methods were performed. The first ELISA assay (ELISA 1) was developed using antibodies acquired against Pulmozyme®. The second ELISA assay (ELISA 2) was developed using antibodies acquired against plant derived DNase I

[0284] The total content of prh DNase I was assessed by indirect sandwich ELISA assays. The antibodies used in ELISA 1 include unfractionated rabbit anti- human DNase I antiserum and chicken yolk IgY fraction. These antibodies were prepared following immunization of rabbits and chickens with commercially available, mammalian cell expressed human DNase I (Pulmozyme®). Unfractionated rabbit anti-human DNase I antiserum was prepared in accordance with a standard polyclonal antibody preparation protocol including four immunizations with 3mg antigen per rabbit, and collection of the serum after the fourth immunization. Anti-human DNase I yolk IgY fraction was prepared in accordance to a standard polyclonal antibody preparation protocol including four immunizations with 2.5mg antigen per chicken, and collection of the yolks after the fourth immunization.

[0285] The antibodies used in ELISA 2 included rabbit anti-prh DNase I and goat-anti-prh DNase I. These antibodies were prepared by immunization of rabbits and goats with prh DNase and affinity purification on a Pulmozyme® column. Thus, the purified antibodies detect/bind the common backbone sequence of Pulmozyme® and prh DNase I. The antibodies were prepared by GenScript USA Inc in accordance to GenScript standard polyclonal antibody preparation protocol including four immunizations with 3mg antigen per rabbit and 8mg antigen per goat, and collection of the serum after the forth immunization.

[0286] Microtiter 96-well plates (Costar cat No. 9018 for ELISA 1 and NUNC cat No. 442404 for ELISA 2) were incubated for overnight at 4°C with 100 µl/well of whole rabbit antiserum 2500-fold diluted (ELISA 1) or 1ug/ml goat anti-prh DNase I antibody (ELISA 2) and diluted in carbonate-bicarbonate buffer pH 9.6 (Sigma cat No. C3041). Plates were then washed four times with wash buffer prepared according to manufacturer's instructions (KPL cat No. 50-63-00), blocked with 300 µl of Blocker Casein™ (Pierce cat No. 37528), incubated for one hour at 25°C and washed again four times with wash buffer. A standard curve was prepared by dilution of standard prh DNase I in dilution buffer (1% (w/v) BSA in phosphate buffered saline) at concentrations ranging from 0.3125 to 20 ng/ml (ELISA 1) or 0.1562 to 10 ng/ml (ELISA 2) at 2-fold series dilutions. Similarly, samples and controls were diluted 100- to 100,000-fold in dilution buffer. One hundred microliters of standards, controls and samples were added in duplicates to the wells of pre-coated and pre-blocked plates, and agitated for two hours at 25° C or incubated overnight at 4° C. Plates were then washed 4 times and incubated for 1.5 hours at 25° C with 100 µl/well of chicken yolk IgY fraction diluted 20,000-fold in the dilution buffer (ELISA 1) or rabbit anti- prh DNase I antibody diluted 10,000-fold in the dilution buffer. Following four additional washing steps, 100 µl of donkey anti-chicken HRP conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., PA; cat No. 703-035-155) (ELISA 1) or donkey anti-rabbit HRP conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., PA; cat No. 711-035-152) (ELISA 2), diluted 5000-fold in dilution buffer, was added and plates were incubated for one hour at 25° C. Next, plates were washed extensively and supplemented with 100 µl TMB solution (Millipore; cat No. ES001). Upon color development reaction was stopped by addition of 100 µl of 10% (v/v) HCl and then read at 450nm. Absorbance was plotted versus the standards concentrations and data were fit to a 4-parameter logistic model by the nonlinear regression method of Marquardt.

[0287] DNase I immunoreactivity in different expression systems was expressed as percent of the activity measured in the apoplast targeted cells (100%)(see FIG. 2).

Enzyme kinetics:



[0288] The kinetics of an enzymatic reaction are typically studied by varying the concentration of substrate and plotting the rate of product formation as a function of substrate concentration (velocity). In the conventional case, this yields a typical hyperbolic Michaelis-Menten curve and a linear reciprocal Lineweaver-Burk plot, from which the kinetic constants of the enzyme can be calculated. For enzyme reactions exhibiting simple Michaelis-Menten kinetics, the Michaelis constant (KM), or the substrate concentration at half maximum velocity (Vmax/2) represents the dissociation constant of the enzyme-substrate (ES) complex or the affinity for substrate. Low KM values indicate higher substrate affinity.

[0289] The kinetic constant (k2), often called kcat, represents the number of substrate molecules converted into product per unit time at a single catalytic site when the enzyme is fully saturated with substrate. kcat is usually the reaction rate limiting step and is calculated by dividing Vmax by the enzyme's concentration. kcat/KM is often thought of as a measure of enzyme efficiency.

[0290] Many enzymes do not behave in a conventional way, their velocity curves rising to a maximum and then declining as the substrate concentration rises, referred to as substrate inhibition, and thought to be the result of binding of more than one substrate molecule to an active site. Substrate inhibition is considered a biologically relevant regulatory mechanism although it is often observed when using artificially high substrate concentrations, in a laboratory setting.

[0291] Kinetic plots for substrate inhibition represent variation of initial velocity as a function of substrate concentration or as double-reciprocal plot of the variation of initial velocity with substrate concentration. Since the same compound acts at the same time as both substrate and inhibitor the double reciprocal plot does not yield a straight line. It may be assumed that inhibitory effect is negligible at low substrate concentration and from an asymptote to the curve in this region KM and Vmax can be estimated.

[0292] Assays of the enzyme kinetics of DNase I activity were performed using the DNaseAlert™ (custom made by IDT) fluorescence-quenched oligonucleotide probe, which has a HEX™ reporter dye (hexachlorofluorescein) on one end and a dark quencher on the other end, and which emits a detectable fluorescent signal upon nuclease degradation. The DNA sequence has been carefully optimized to react with a wide variety of nucleases; it contains domains that will react with single-stranded endonucleases, certain single-stranded exonucleases, and double-stranded nucleases. The intact substrate has little or no fluorescence, but when cleaved by a DNase, the hydrolyzed substrate fluoresces pink (536 nm or UV excitation, 556 nm emission) when cleaved, and can be detected visually or using a fluorometer. Plots of DNase I initial velocity versus substrate concentration display a velocity curve that rises to a maximum and then declines as the substrate concentration increases (Fig 10A). This pattern can characterize a typical substrate inhibition behavior. The double reciprocal plot allows the extraction of KM and Vmax values by abscissa intercept with an asymptote to the high substrate region (approaching the origin), where the inhibitory effect predominates (Fig 10B).

DNaseAlert™ based kinetic assay



[0293] DNaseAlert™ substrate (IDT; custom made) was diluted in Activity Buffer (25mM HEPES-NaOH, 4mM CaCl2, 4mM MgCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5) at concentrations ranging from 1 to 40 µM. Ten microliters of substrate was divided in duplicates into a black 96-well plate (Greiner cat No. 655900) and the reaction was initiated by rapid addition of 10 ul DNase I to a final concentration of 50 ng/mL. The plate was immediately incubated in a fluorometer and real-time data was collected (535 nm excitation, 565 nm emission) at 30 seconds intervals for 3 minutes. Initial velocities were extracted by using fluorescent product standard curve. Product standard curve was prepared by dilution of 6-HEX (AnaSpec) in activity buffer at concentrations ranging from 7 to 250 nM at 1.67-fold series dilutions. Fluorescence units were transformed to concentration values (µM) by applying the calibration curve formula (y=ax+b when y=FU and a=nM). A double reciprocal plot of initial velocity versus substrate concentration (at 2-15 µM) allows the extraction of KM and Vmax values by abscissa intercept with an asymptote to the high substrate region (Fig 10B).

Specific activity



[0294] Specific activity of human DNase I was evaluated using the DNaseAlert™ fluorescence-quenched oligonucleotide probe (supplied by Ambion, cat #AM1970, manufactured by IDT) that emits a fluorescent signal upon nuclease degradation. One unit (U) of DNase is defined as the amount of enzyme that releases 1 µmol of fluorescent product per minute at 22°C. The value of specific activity (U/mg) is calculated by dividing the value of total activity (U) by the amount of enzyme used in the reaction. Enzyme concentration was determined by absorbance at 280nm, based on extinction coefficient. The calculated extinction coefficient of phr DNase I is 1.45 g/L.

[0295] 2nmol of DNaseAlert™ substrate (Ambion, cat #AM1970) was resuspended with 1 mL nuclease free water. Prh DNase I and Pulmozyme® samples were diluted 10,000-40,000-fold in activity buffer (25mM HEPES-NaOH, 4mM CaCl2, 4mM MgCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5). 80 uL of activity buffer was divided in duplicates to a black 96-well plate (Greiner cat No. 655900). Ten microliters of substrate was then added and the reaction was initiated by rapid addition of 10ul Rh DNase I or Pulmozyme®. The plate was immediately incubated in a fluorometer and real-time data was collected (535 nm excitation, 565 nm emission) at 30-second intervals for 3 minutes. Product standard curve was prepared by dilution of 6-HEX (AnaSpec cat # 81019) in activity buffer at concentrations ranging from 7 to 250 nM at 1.67-fold series dilutions. Fluorescence units were transformed to concentration values (µM) by applying the calibration curve formula (y=ax+b when y=FU and a=nM). 1 U is defined as the amount of enzyme that releases 1 µmol of fluorescent product per minute at 22°C. The value of specific activity (U/mg) was calculated by dividing the value of total activity (U) by the amount of enzyme used in the reaction.

Results:


Plant Expressed rh DNase I has superior enzyme kinetics compared to mammalian cell expressed human DNase I



[0296] Figure 10A shows representative substrate inhibition kinetic plots of plant expressed rh DNase I and Pulmozyme®. Initial velocity versus substrate concentration plots yielded a curve that is typical for a substrate inhibition pattern (see Table VI) for both enzymes. The KM, and Vmax values were calculated from asymptote equation plotted in a region of low substrate concentrations (2-15 µM) in double reciprocal plots (Figure 10B). kcat values of phr DNase I and Pulmozyme® were calculated by dividing Vmax with enzyme's concentration (Table VI), further indicating that prh DNase I and Pulmozyme® enzyme kinetics exhibit characteristics of the substrate inhibition kinetic model.

[0297] However, it is evident that although both enzymes comprise the same DNase I amino acid sequence, prh DNase I exhibits improved kinetic properties (greater substrate affinity and greater enzyme velocity) compared to Pulmozyme®. Without wishing to adhere to a single hypothesis, this may be due to unique post-translational modifications of the plant-expressed rh DNase I generated as a result of expression of the nucleic acid construct of the present invention in plant, rather than mammalian expression systems. Three batches of DNase extracted from cultured cells were analyzed and have shown similar characteristics. Further analysis of batches of DNase extracted from cells sampled from different lines of transformed BY2 cells at different times confirmed the reproducible, favorable kinetic characteristics of the plant expressed rh DNase as compared to Pulmozyme®.
Table VI - Kinetic parameters of plant expressed rh DNase I vs. Pulmozyme®
DNase IKM (µM)Vmax (µM/min)kcat (sec-1)kcat /KM (sec-1µM-1)
Plant-expressed rh DNase I 24.3 0.58 6.24 0.26
Pulmozyme® 60.9 0.44 4.76 0.08
Table VIa - Kinetic parameters of 3 plant expressed rh DNase I batches
prhDNase I lineKM (µM)Vmax (µM/min)kcat (sec-1)kcat /KM (sec-1µM-1)
1 16.77 0.37 5.58 0.33
2 12.54 0.36 5.44 0.43
2 12.07 0.35 5.29 0.44

Specific activity of plant-expressed rh DNase I



[0298] Specific activity values of plant-expressed rh DNase I and mammalian cell expressed DNase I (Pulmozyme®) are presented in Table VII. Significantly, the specific activity of plant-expressed rh DNase I, as assayed with the DNaseAlert™ fluorescence-quenched oligonucleotide probe is about 3-fold higher than that of the Pulmozyme®. This difference in plant-expressed rh DNase I and Pulmozyme® specific activity reflects the improved kinetic properties of plant-expressed rh DNase I compared to Pulmozyme® (see Figures 10A and 10B and Table VI above).
Table VII - Specific activity of plant-expressed rh DNase I vs. Pulmozyme
DNase ISpecific activity (U/mg)
Plant-expressed rh DNase I 0.258 ± 0.055
Pulmozyme® 0.091 ± 0.018


[0299] Thus, plant-expressed prh DNase I is fully active catalytically, towards conventional DNase I substrates, exhibits significantly superior enzyme kinetics, and possesses a higher specific activity, consistent with the improved enzyme kinetics, when compared to that of the commercially available, clinical standard mammalian cell expressed recombinant DNase I.

EXAMPLE 4- SUPERIOR RESISTANCE OF PLANT-EXPRESSED rh DNASE I to ACTIN INHIBITION



[0300] Analysis of the composition of CF sputum reveals large quantities (3 to 14 mg/ml) of DNA and actin (0.06 to 5 mg/ml) released by necrosing neutrophils after their recruitment into the airways during response to infection. In addition to hydrolysis of deoxynucleic acid, DNase I can depolymerize filamentous actin (F-actin). Monomeric globular actin (G-actin) is a potent inhibitor (Ki 1 nM) of DNase I enzymatic activity, potentially influencing the effectiveness of inhaled DNase I in CF lungs.

[0301] To evaluate G-actin inhibitory effect on DNase I activity, an IC50 assay (half maximal inhibitory concentration) was developed, applying Methyl Green enzymatic activity assay in the presence of elevated concentrations of human non-muscle actin (Cytoskeleton; cat No. APHL99).

[0302] One hundred microliters of actin/DNase-I mixture was divided in duplicates to a 96-well plate (NUNC cat No. 442404) containing 100ul of DNA-methyl green substrate. Human non-muscle actin (Cytoskeleton; cat No. APHL99) was diluted in activity buffer (25mM HEPES-NaOH, 4mM CaCl2, 4mM MgCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5) containing 0.1mM ATP (Sigma cat No. A26209) to reach concentrations ranging from 100 to 0.1 ug/mL, at 2-fold series dilutions. Prh DNase I and Pulmozyme® (control) were diluted to reach concentration of 100 ng/mL. Each plate's content was then mixed thoroughly, plates were read at 620nm, sealed and incubated for 4 hr at 37 °C and read again (620nm). The change in absorbance was plotted versus actin concentrations and IC50 parameters were calculated by non-linear fit using GraFit software (Erithacus Software, UK).

Results: Plant expressed rh DNase I exhibits improved resistance to actin inhibition



[0303] Figure 11 shows plots describing plant expressed rh DNase I and Pulmozyme® catalytic activity (expressed as ΔOD620nm) in the presence of increasing concentrations of human G-actin. The plot of the change in absorbance versus actin concentration yields hyperbolic curves allowing the extraction of IC50 (Table VIII, below). Although both enzymes have the same amino acid sequence, the plant expressed rh DNase I displays greater resistance to inhibition by human actin than that of the commercial, mammalian cell expressed DNase (Pulmozyme®). Three batches of DNase extracted from cultured cells were analyzed and have shown similar characteristics. Yet further, comparison of resistance/susceptibility to actin inhibition of the plant expressed rhDNase I sampled from different lines of transformed BY2 cells expressing the recombinant enzyme (see Table VIIIa below) confirmed the reproducible improved resistance to actin inhibition. Without wishing to be limited to a single hypothesis, one explanation for this increased resistance to actin inhibition could be the result of the improved affinity of the plant expressed enzyme for its DNA substrate, as compared to that of the mammalian expressed enzyme, or actual reduced affinity of the prhDNase I for actin monomers, or both.
Table VIII -
IC50 of plant expressed rh DNase I vs. Pulmozyme®
DNase IIC50 (µg/ml)
Prh DNase I 1.8191 ± 0.1003
Pulmozyme® 0.6870 ± 0.0204
Table VIIIa -
IC50 of plant expressed rh DNase I vs. Pulmozyme®
DNase IIC50 (µg/ml)
Prh DNase I batch 1 1.7067 ± 0.0568
Prh DNase I batch 2 1.8453 ± 0.0429
Pulmozyme® 0.5458 ± 0.0115

EXAMPLE 5- EFFECT OF rh DNASE I ON THE RHEOLOGICAL PROPERTIES OF SPUTUM



[0304] Mucus is defined as the heterogeneous, adhesive, viscoelastic gel produced by goblet cells and submucosal glands. At the chemical level, mucus is an integrated structure of biopolymers. Its physical behavior is complex (non-Newtonian), with highly variable properties that are between those of a viscous liquid and an elastic solid. Characterization of the physical properties of mucus largely focuses on two properties: (i) viscous modulus, also termed loss modulus (G", expressed in pascal-seconds-Pa.s), which is the extent to which the gel resists the tendency to flow, and (ii) elastic modulus, also termed storage modulus (G', expressed in pascals- seconds-Pa.s), which expresses the tendency for the gel to recover its original shape following stress-induced deformation. Together, these properties describe the rheology of complex biological fluids. The phase angle or loss tangent value δ, calculated from the inverse tangent of G"/ G' (tan δ □= G"/ G') is also a common parameter for characterizing mucus, reflecting the overall elastic or viscous nature of the sample and used to quantify the extent of elastic behaviour of material. A phase angle close to zero indicates strongly elastic behaviour, as opposed to 90°, which indicates purely viscous behaviour. A phase angle of 45° is considered the "cross over" value between G" and G'.

CF Sputum collection, storage and sample treatment



[0305] CF sputum samples were collected from patients with severe lung disease attending the Cystic Fibrosis Centers. Sputum was directly expectorated into a sterile hermetically sealed container and transported on ice to laboratories equipped for rheological characterization. Saliva was removed and each sputum sample was homogenized gently and divided into 200-300 mg aliquots and stored at -70°C until analyzed. Frozen samples were thawed at room temperature before analysis, as freezing of the sputum samples followed by a single thawing step was shown to afford accurate and reproducible analysis of sputum rheology, similar to those of the fresh sample before freezing.

[0306] In order to ensure that the sputum is free of exogenous DNase I activity (e.g. Pulmozyme®), sputum samples were collected 12-24 hours after treatment with Pulmozyme® aerosol, as it has been reported that inhaled aerosol DNase I is cleared from the sputum from patients as soon as two hours.

Study design



[0307] Each sputum aliquot was incubated for 30 min at 37°C with DNase I formulation buffer (1mM CaCl2, 150mM NaCl, pH 6.1-6.5) containing either prh DNase I or Pulmozyme® (final concentration 0.2, 2, 5, 10 and 20 ug/gr sputum). Control samples were treated with DNase I formulation buffer only.

[0308] Four percent (vol/wt) of drug or control were added to the sputum sample. Following incubation, rheological properties of sputum samples were immediately measured.

Sputum rheology assay



[0309] Rheological properties of the sputum samples were determined using a controlled stress rheometer (HAAKE RheoStress 1, Thermo Fisher Scientific GmbH, Karlsruhe, Germany). The measurements were conducted at 20 °C using two techniques: a time-sweep (Figs. 12A-12D and 13A-13D) and a stress sweep (Figs 18A-18D and 19A-19D).

[0310] Time sweep measurements were performed using a 35 mm cone plate set-up. The angle between the cone and the plate was 0.5° and the sample volume required was 150 ul.Nondestructive oscillatory stress was applied to the sample and the elastic (G') and viscous (G") modulus were recorded verus time. To avoid disruption of the weak biopolymer network in the sputum due to the oscillation forces, the measurements were performed in the linear viscoelastic region at a constant frequency of 1 Hz with a stress of 0.10 Pascals (Pa).

[0311] Stress sweep measurements were performed using 20mm sandblasted parallel plate geometry. The sputum samples (200 ul) were loaded onto the rheometer with a gap width of 0.5mm. A stress sweep was performed from 0.1 to 100 Pa at a constant frequency of 1 Hz and the elastic modulus, (G'), viscous modulus (G") and phase angle (δ) were measured. The applied stress in which G' and G" cross over (or phase angle reaches 45°) is the stress in which the sample begins to act more liquid-like than solid-like. At this point stress values were recorded and compared between DNase I treated and untreated samples.

[0312] Rheological parameters were determined using RheoWin 4 (HAAKE, Thermo Fisher Scientific GmbH, Karlsruhe, Germany). Before measurements, sputum samples were loaded into the rheometer plate and equilibrated for 30 seconds to allow relaxation to the original gel structure. In order to slow down the dehydration of the sputum, a solvent trap was used. Experiments were performed on at least two sputum fractions taken from each sputum sample and the data were averaged.

Measurements of total DNA content in sputum



[0313] Sputum DNA content was determined by the modified aminobenzoic acid (DABA) assay. Salmon sperm DNA (Sigma #D1626, ∼2mg/mL) and sputum samples (∼100mg) were diluted 10-fold in a dilution buffer (25mM HEPES-NaOH, 4mM CaCl2, 4mM MgCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5) and incubated at 60°C for 1 hour. Samples were repeatedly vortexed to allow sputum disintegration. DNA concentration of the diluted salmon sperm sample was then measured (NanoDrop 2000, Thermo Fisher Scientific) and standard curve was prepared by dilution of the salmon sperm sample in the dilution buffer at concentrations ranging from 3.13 to 200 µg/ml at 2-fold series dilutions. Similarly, sputum samples were further diluted at 2-fold series dilution up to 1280-fold. Next, fifty microlitres of standards and samples was added in duplicates to a black 96-well plate (Greiner cat No. 655900) and incubated with 50 µL of 20% 3, 5-diaminobenzoic acid (TCI Europe cat No. D0079) at 60°C for 1 hour. Fifty (50) ul of 5N HCl was then added to stop the reaction and fluorescence was measured by fluorometer (390 nm excitation /530 nm emission). Fluorescence units were plotted versus standard DNA concentrations and the data were fit to a 4-parameter logistic model by the nonlinear regression method of Marquardt. DNA concentration in sputum was then interpolated.

Magnesium chloride and rh DNase I effect on rheological properties of sputum



[0314] Magnesium ion is a cofactor of DNase I, hence promoting DNA hydrolysis. Studies suggest that increasing magnesium concentration in the airway surface liquid by aerosolisation of magnesium solutions or oral magnesium supplements could improve the removal of highly viscous mucus in chronic lung disease by improving DNase activity. Furthermore, it was suggested that magnesium indirectly triggers the activity of rhDNase I in CF sputum by promoting polymerization of G-actin into F-actin, hence, reducing inhibition of DNase-I by G-actin. Consequently, magnesium supplement may overcome poor response to rhDNase treatment in non-responding CF patients exhibiting low levels of magnesium in their sputum.

Magnesium sulfate and rh DNase I effect on catalytic activity of rh DNase I



[0315] Several trials conducted with inhaled magnesium sulfate in patients with acute asthma have shown that magnesium sulfate inhalation is tolerable. Magnesium ions, required for DNase I activity, can therefore be supplemented to rh DNase I formulation as magnesium sulfate. To verify that magnesium sulfate does not produce an inhibitory effect on rh DNase I activity a methyl green activity assay was applied in the presence of elevated concentrations of magnesium sulfate and compared to magnesium chloride effect.

[0316] One hundred microliters of plant expressed rh DNase I was divided in duplicates to a 96-well plate (NUNC cat No. 442404) containing 100ul of DNA-methyl green substrate to reach concentration of 100 ng/mL. Reaction was prepared in a modified activity buffer (25mM HEPES-NaOH, 4mM CaCl2, 0.1% BSA, 0.05% Tween-20, pH 7.5) containing elevated concentration of magnesium sulfate or magnesium chloride (0.5-100mM). The plate's contents were then mixed thoroughly, and the plates were read at 620nm, sealed and incubated for 3 hr at 37°C and re-read (620nm). Change in absorbance was plotted versus concentrations of magnesium sulfate or magnesium chloride added.

Results:


Plant expressed rh DNase I is highly effective in reducing rheological properties of sputum of CF patients.



[0317] The rheological properties of sputum samples were measured following incubation of CF sputum with DNase I formulation buffer or different concentrations of plant expressed rh DNase I and Pulmozyme®, diluted in DNase I formulation buffer (final concentration 0.2, 2, 5, 10 and 20 ug/gr sputum) (see Figures 12A- 12D ; 13A-13D; 18A-18D and 19A-19D). DNase I concentrations correspond to the concentrations detected 15 min after aerosolization of Pulmozyme® recommended therapeutic dose (mean value of 2.9 µg/ml mucus).

[0318] Time sweep measurement (Figures 12A-12D and 13A-13D) clearly revealed that incubation with the plant expressed rh DNase I significantly reduced sputum elastic modulus (Figure 12A-12D) and viscous modulus (Figure 13A-13D), in a concentration-dependent manner, and with a consistently greater efficiency (per ug DNase I; Figure 12A) than the clinically approved mammalian cell-expressed Pulmozyme®. Even greater expression of the improved rheological properties of the prh DNase I-treated sputa was observed when the reduction elastic and viscous modulus following incubation with prh DNase I was expressed as percent change compared to the untreated control (see Figures 14A and 14B). In addition, stress sweep measurements (see Figures 18A-18D and 19A-19D) reveals that the plant expressed rh DNase I disrupts the sputa elastic structure (the internal network of the sputa) in a dose- dependent manner and with greater efficiency than the clinically approved mammalian cell-expressed Pulmozyme®. The improved efficacy of the prh DNase I is consistent with the improved enzyme kinetics and the higher specific activity of the prh DNase I, compared to Pulmozyme® (see Examples 2 and 3 above).

Synergistic reduction of CF sputum rheological properties with plant expressed rh DNase I and magnesium



[0319] Time sweep measurements evaluating rheological properties of CF sputum were performed following incubation of sputum samples with plant expressed rh DNase I protein, diluted in DNase I formulation buffer (final concentration of 2ug/gr sputum), or DNase I formulation buffer (control), in the presence or absence of 25, 50 and 100mM magnesium chloride, prepared in DNase I formulation buffer. Figures 15 and 16 clearly indicate that incubation of sputum with 25-100 mM magnesium chloride (grey bars), or prhDNase alone ("0" MgCl2 in Figures 15 and 16) results in reduction in both the sputum elastic (Figure 15) and viscous (Figure 16) modulus. When the sputum is incubated with both magnesium chloride and plant expressed rh DNase I, (hatched bars, 25-100 mM MgCl2), a significant synergistic reduction can be observed following a threshold of added Mg (in this experiment 100mM Mg) in both sputum rheological properties assayed [elastic (Figure 15) and viscous (Figure 16) modulus].

[0320] Stress sweep measurements were performed following incubation of sputum samples with plant expressed rh DNase I protein, diluted in DNase I formulation buffer, or DNase I formulation buffer (control), in the presence or absence of 100mM magnesium sulfate, prepared in DNase I formulation buffer (see FIGs. 20A-20C). Taken together, these results demonstrate that incubation of CF sputum with both magnesium sulfate (Figure 20A-20C, grey bars) and prhDNase (Figure 20A-20C, dark bars) results in a synergistically greater and more significant disruption in sputum elastic structure than incubation with prhDNase or magnesium sulfate alone. This is particularly evident in patient sample A, figure 20A, where reduction in the stress sweep values for sputum sample by prhDNase was detected primarily when magnesium sulfate is added.

Magnesium salts do not inhibit plant expressed rh DNase I activity



[0321] When catalytic activity of plant expressed rh DNase I was assayed in the presence of different concentrations of magnesium sulfate and magnesium chloride, a dose-dependent improvement of DNA hydrolytic activity was observed with concentrations between 0.5 to 10 mM magnesium, with no difference between activity in the presence of chloride salt (MgCl2) or sulphate salt (MgSO4) (Figure 16). DNA hydrolytic activity of the plant expressed rh DNase I remained optimal with both magnesium sulfate and magnesium chloride at concentration of 10-50 mM, with only a slight depression of activity with magnesium sulphate, compared to magnesium chloride, at 100mM. Thus, neither the chloride salt (MgCl2) or sulphate salt (MgSO4) significantly impairs DNA hydrolytic activity of the plant expressed rh DNase I over a wide range of concentrations.

[0322] Taken together, these results show that incubation with plant expressed rh DNase I improves the rheological properties of CF sputum. The dose dependent reduction in sputum rheological properties achieved with prh DNase I is more dramatic than that observed with mammalian cell expressed DNae I (Pulmozyme®). Yet further, it was observed that addition of magnesium chloride can improve the effect of plant expressed rh DNase I on the rheological properties of sputum, and that the DNA hydrolytic activity of plant expressed rh DNase I is enhanced by both magnesium chloride and magnesium sulphate over a wide range of concentrations. Thus, magnesium sulfate can be supplemented to prh DNase I to improve the prh DNase I's mucolytic activity in-vivo.

[0323] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the broad scope of the appended claims.

SEQUENCE LISTING



[0324] 

<110> Protalix Ltd. Shaaltiel, Yoseph Hanania, Uri Kizhner, Tali Matiuhin, Yulia Fux, Liat Shulman, Avidor

<120> DNASE I POLYPEPTIDES, POLYNUCLEOTIDES ENCODING SAME, METHODS OF PRODUCING DNASE I AND USES THEREOF IN THERAPY

<130> 55619

<150> US 61/593,676
<151> 2012-02-01

<150> US 61/593,788
<151> 2012-02-01

<150> US 61/593,350
<151> 2012-02-01

<160> 291

<170> PatentIn version 3.5

<210> 1
<211> 293
<212> PRT
<213> Artificial sequence

<220>
<223> Plant recombinant human DNase I

<400> 1



<210> 2
<211> 282
<212> PRT
<213> Homo sapiens

<400> 2





<210> 3
<211> 22
<212> PRT
<213> Homo sapiens

<400> 3

<210> 4
<211> 33
<212> PRT
<213> Artificial sequence

<220>
<223> Arabidopsis derived ABPI endoplasmic reticulum targeting signal peptide

<400> 4

<210> 5
<211> 261
<212> PRT
<213> Artificial sequence

<220>
<223> Putative amino acid sequence of the purified, plant expressed rhDNase I

<400> 5



<210> 6
<211> 260
<212> PRT
<213> Homo sapiens

<400> 6



<210> 7
<211> 261
<212> PRT
<213> Artificial sequence

<220>
<223> Mature plant recombinant human DNaseI + G

<400> 7



<210> 8
<211> 293
<212> PRT
<213> Artificial sequence

<220>
<223> Plant recombinant human DNase I

<400> 8



<210> 9
<211> 786
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence of the recombinant DNase I without the leader sequence

<400> 9



<210> 10
<211> 99
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence of polypeptide of ABPI leader signal

<400> 10

<210> 11
<211> 849
<212> DNA
<213> Homo sapiens

<400> 11



<210> 12
<211> 885
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence of rhDNase I (ABPI+DNase I)

<400> 12

<210> 13
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> Endoplasmic reticulum retention signal SEKDEL

<400> 13

<210> 14
<211> 18
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence of SEKDEL (ER retention signal)

<400> 14
tctgagaagg atgagctt   18

<210> 15
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> Vacuole targeting signal peptide GLLVDTM

<400> 15

<210> 16
<211> 21
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence for vacuole targeting signal peptide GLLVDTM

<400> 16
ggacttcttg ttgatactat g   21

<210> 17
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 17

<210> 18
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 18

<210> 19
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 19

<210> 20
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 20

<210> 21
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 21

<210> 22
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 22

<210> 23
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 23

<210> 24
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 24

<210> 25
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 25

<210> 26
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 26

<210> 27
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 27

<210> 28
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 28

<210> 29
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 29

<210> 30
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 30

<210> 31
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 31

<210> 32
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 32

<210> 33
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 33

<210> 34
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 34

<210> 35
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 35

<210> 36
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 36

<210> 37
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 37

<210> 38
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 38

<210> 39
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 39

<210> 40
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 40

<210> 41
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 41

<210> 42
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 42

<210> 43
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 43

<210> 44
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 44

<210> 45
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 45

<210> 46
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 46

<210> 47
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 47

<210> 48
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 48

<210> 49
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 49

<210> 50
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 50

<210> 51
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 51

<210> 52
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 52

<210> 53
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 53

<210> 54
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 54

<210> 55
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 55

<210> 56
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 56

<210> 57
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 57

<210> 58
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 58

<210> 59
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 59

<210> 60
<211> 32
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 60

<210> 61
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 61

<210> 62
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 62

<210> 63
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 63

<210> 64
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 64

<210> 65
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 65

<210> 66
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 66

<210> 67
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 67

<210> 68
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 68

<210> 69
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 69

<210> 70
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 70

<210> 71
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 71

<210> 72
<211> 27
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 72

<210> 73
<211> 29
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 73

<210> 74
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 74

<210> 75
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 75

<210> 76
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 76

<210> 77
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 77

<210> 78
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 78

<210> 79
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 79

<210> 80
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 80

<210> 81
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 81

<210> 82
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 82

<210> 83
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 83

<210> 84
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 84

<210> 85
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 85

<210> 86
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 86

<210> 87
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 87

<210> 88
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 88

<210> 89
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 89

<210> 90
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 90

<210> 91
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 91

<210> 92
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 92

<210> 93
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 93

<210> 94
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 94

<210> 95
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 95



<210> 96
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 96

<210> 97
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 97

<210> 98
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 98

<210> 99
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 99

<210> 100
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 100

<210> 101
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 101

<210> 102
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 102

<210> 103
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 103

<210> 104
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 104

<210> 105
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 105

<210> 106
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 106

<210> 107
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 107

<210> 108
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 108

<210> 109
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 109

<210> 110
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 110

<210> 111
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 111

<210> 112
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 112

<210> 113
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 113

<210> 114
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 114

<210> 115
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 115

<210> 116
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 116

<210> 117
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 117

<210> 118
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 118

<210> 119
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 119

<210> 120
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 120

<210> 121
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 121

<210> 122
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 122

<210> 123
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 123

<210> 124
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 124

<210> 125
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 125

<210> 126
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 126

<210> 127
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 127



<210> 128
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 128

<210> 129
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 129

<210> 130
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 130

<210> 131
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 131

<210> 132
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 132

<210> 133
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 133

<210> 134
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 134

<210> 135
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 135

<210> 136
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 136

<210> 137
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 137

<210> 138
<211> 22
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 138

<210> 139
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 139

<210> 140
<211> 24
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 140

<210> 141
<211> 25
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 141

<210> 142
<211> 31
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 142



<210> 143
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 143

<210> 144
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 144

<210> 145
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 145

<210> 146
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 146

<210> 147
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 147

<210> 148
<211> 28
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 148

<210> 149
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 149

<210> 150
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 150

<210> 151
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 151

<210> 152
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 152

<210> 153
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 153

<210> 154
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 154

<210> 155
<211> 24
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 155

<210> 156
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 156

<210> 157
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 157

<210> 158
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 158

<210> 159
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 159

<210> 160
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 160

<210> 161
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 161

<210> 162
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 162

<210> 163
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 163

<210> 164
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 164

<210> 165
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 165

<210> 166
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 166

<210> 167
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 167

<210> 168
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 168

<210> 169
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 169

<210> 170
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 170

<210> 171
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 171

<210> 172
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 172

<210> 173
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 173

<210> 174
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 174

<210> 175
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 175

<210> 176
<211> 28
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 176

<210> 177
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 177

<210> 178
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 178

<210> 179
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 179

<210> 180
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 180



<210> 181
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 181

<210> 182
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 182

<210> 183
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 183

<210> 184
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 184



<210> 185
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 185

<210> 186
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 186

<210> 187
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 187

<210> 188
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 188

<210> 189
<211> 14
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 189

<210> 190
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 190

<210> 191
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 191

<210> 192
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 192

<210> 193
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 193

<210> 194
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 194

<210> 195
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 195

<210> 196
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 196

<210> 197
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 197

<210> 198
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 198

<210> 199
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 199

<210> 200
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 200

<210> 201
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 201

<210> 202
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 202

<210> 203
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 203

<210> 204
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 204

<210> 205
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 205

<210> 206
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 206

<210> 207
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 207

<210> 208
<211> 26
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 208

<210> 209
<211> 28
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 209

<210> 210
<211> 27
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 210

<210> 211
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 211

<210> 212
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 212

<210> 213
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 213

<210> 214
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 214

<210> 215
<211> 24
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 215

<210> 216
<211> 26
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 216

<210> 217
<211> 22
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 217

<210> 218
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 218

<210> 219
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 219



<210> 220
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 220

<210> 221
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 221

<210> 222
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 222

<210> 223
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 223

<210> 224
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 224

<210> 225
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 225

<210> 226
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 226

<210> 227
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 227

<210> 228
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 228

<210> 229
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 229

<210> 230
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 230

<210> 231
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 231

<210> 232
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 232

<210> 233
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 233

<210> 234
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 234

<210> 235
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 235

<210> 236
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 236

<210> 237
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 237

<210> 238
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 238

<210> 239
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 239

<210> 240
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 240

<210> 241
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 241

<210> 242
<211> 12
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 242

<210> 243
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 243

<210> 244
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 244

<210> 245
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 245

<210> 246
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 246

<210> 247
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 247

<210> 248
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 248

<210> 249
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 249

<210> 250
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 250

<210> 251
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 251

<210> 252
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 252

<210> 253
<211> 38
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 253

<210> 254
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 254

<210> 255
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 255

<210> 256
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 256

<210> 257
<211> 27
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 257

<210> 258
<211> 26
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 258

<210> 259
<211> 25
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 259

<210> 260
<211> 24
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 260

<210> 261
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 261

<210> 262
<211> 8
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 262

<210> 263
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 263

<210> 264
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 264

<210> 265
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 265

<210> 266
<211> 18
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 266

<210> 267
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 267

<210> 268
<211> 20
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 268

<210> 269
<211> 21
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 269

<210> 270
<211> 19
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 270

<210> 271
<211> 17
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 271

<210> 272
<211> 15
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 272

<210> 273
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 273

<210> 274
<211> 11
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 274

<210> 275
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 275

<210> 276
<211> 7
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and Chymotrypsin of the plant-expressed rh DNase I

<400> 276

<210> 277
<211> 789
<212> DNA
<213> Artificial sequence

<220>
<223> Coding sequence of the recombinant DNase I with an additional N' Gly residue (DNase I + G)

<400> 277

<210> 278
<211> 13
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 278

<210> 279
<211> 16
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 279



<210> 280
<211> 10
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 280

<210> 281
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 281

<210> 282
<211> 23
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 282

<210> 283
<211> 27
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 283

<210> 284
<211> 32
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 284

<210> 285
<211> 38
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 285

<210> 286
<211> 6
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 286

<210> 287
<211> 4
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 287

<210> 288
<211> 5
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 288

<210> 289
<211> 31
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 289

<210> 290
<211> 9
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 290



<210> 291
<211> 38
<212> PRT
<213> Artificial sequence

<220>
<223> A peptide identified following digestion with Trypsin and PNGaseA of the plant-expressed rh DNase I

<400> 291




Claims

1. An isolated, plant expressed recombinant human DNase I protein consisting of a plant expressed recombinant human DNase I protein having a Glycine residue as the N-terminal amino acid, said recombinant human DNase I protein having reduced susceptibility to actin inhibition of endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I, wherein said plant expressed recombinant human DNase I protein has the amino acid sequence as set forth in SEQ ID NO: 5 and wherein said plant expressed recombinant human DNase I protein has at least one core α-(1,3) fucose.
 
2. A stably or transiently transformed plant cell recombinantly expressing the human DNase I protein of claim 1.
 
3. A method of producing the recombinant human DNase I protein of claim 1, comprising:

providing the stably or transiently transformed plant cell of claim 2; and

growing said plant cell so as to produce said recombinant human DNase I protein and optionally further comprising isolating said recombinant human DNase I protein from said plant cell.


 
4. The human DNase protein of claim 1, having lower Michaelis constant (Km) and greater reaction velocity (Vmax) for endonuclease activity as compared with that of mammalian cell expressed human recombinant DNase I.
 
5. A pharmaceutical composition comprising, as an active ingredient, the isolated human DNase I protein of claim 1 or 4 and a pharmaceutically acceptable carrier.
 
6. The pharmaceutical composition of claim 5, wherein said isolated human DNase I protein is at least 90-95% pure human DNase I protein or wherein the pharmaceutical composition further comprises plant beta-acetylhexosaminidase enzyme protein.
 
7. The pharmaceutical composition of claim 5 for use in treating a DNase-I related disease or condition in a subject in need thereof.
 
8. The pharmaceutical composition of claim 7 for use in treating a DNase-I related disease or condition in a subject in need thereof by reducing the viscous modulus of sputum.
 
9. The pharmaceutical composition of claim 7 for the use in treating a DNase-1 related disease or condition in a subject in need thereof by reducing the elastic modulus of sputum.
 
10. The pharmaceutical composition of claim 5 for use in treating cystic fibrosis (CF) in a subject in need thereof.
 
11. The pharmaceutical composition of claim 5 for use in treating chronic obstructive pulmonary disease (COPD) in a subject in need thereof.
 


Ansprüche

1. Isoliertes, pflanzen-exprimiertes, rekombinantes humanes DNase-1-Protein, bestehend aus einem pflanzen-exprimierten, rekombinanten humanen DNase-I-Protein mit einem Glycinrest als N-terminale Aminosäure, wobei dieses rekombinante humane DNase-1-Protein eine verringerte Suszeptibilität für eine Aktinhemmung der EndonukleaseAktivität verglichen mit jener einer Säugerzelle-exprimierten, humanen rekombinanten DNase-I hat, wobei dieses pflanzen-exprimierte, rekombinante humane DNase-1-Protein die Aminosäuresequenz wie in SEQ ID Nr. 5 hat und wobei dieses pflanzen-exprimierte, rekombinante humane DNase-I-Protein zumindest eine Kern α-(1,3)-Fucose hat.
 
2. Stabil oder transiert transformierte Pflanzenzelle, die das humane DNase-1-Protein nach Anspruch 1 rekombinant exprimiert.
 
3. Verfahren zum Herstellen des rekombinanten, humanen DNase-1-Proteins nach Anspruch 1, umfassend:

Bereitstellen der stabil oder transient transformierten Pflanzenzelle nach Anspruch 2; und

Züchten dieser Pflanzenzelle, um das pflanzen-exprimierte, rekombinante humane DNase-1-Protein herzustellen, und optional ferner umfassend ein Isolieren des rekombinanten, humanen DNase-I-Proteins von der Pflanzenzelle.


 
4. Humanes DNase-1-Protein nach Anspruch 1, mit einer geringeren Michaeliskonstante (Km) und einer größeren Reaktionsgeschwindigkeit (Vmax) für Endonukleaseaktivität verglichen mit jenen der Säugerzelle-expremierten, humanen rekombinanten DNase I.
 
5. Pharmazeutische Zusammensetzung, umfassend, als einen aktiven Inhaltsstoff, das isolierte, humane, DNase-I-Protein nach Anspruch 1 oder 4, und einen pharmazeutisch akzeptablen Träger.
 
6. Pharmazeutische Zusammensetzung nach Anspruch 5, wobei das isolierte, humane DNase-I-Protein ein zumindest 90 - 95 % reines humanes DNase-I-Protein ist, oder wobei die pharmazeutische Zusammensetzung ferner pflanzliches Beta-AcetylHexosaminidase-Enzymprotein aufweist.
 
7. Pharmazeutische Zusammensetzung nach Anspruch 5 zur Verwendung bei der Behandlung einer/s DNase-I-bezogenen Krankheit oder Zustands bei einem Subjekt, das diese benötigt.
 
8. Pharmazeutische Zusammensetzung nach Anspruch 7 zur Verwendung bei der Behandlung einer/s DNase-I-bezogenen Krankheit oder Zustands bei einem Subjekt, das diese benötigt, durch Verringern des Viskositätsmoduls eines Sputums.
 
9. Pharmazeutische Zusammensetzung nach Anspruch 7 zur Verwendung bei der Behandlung einer/s DNase-1-bezogenen Krankheit oder Zustands bei einem Subjekt, das diese benötigt, durch Verringern des Elastizitätsmoduls des Sputums.
 
10. Pharmazeutische Zusammensetzung nach Anspruch 5 zur Verwendung bei der Behandlung einer zystischen Fibrose (CF) bei einem Subjekt, das diese benötigt.
 
11. Pharmazeutische Zusammensetzung nach Anspruch 5 zur Verwendung bei der Behandlung von chronisch obstruktiver Lungenkrankheit (COPD) in einem Subjekt, das diese benötigt.
 


Revendications

1. Protéine DNase I humaine, recombinante, exprimée par une plante et isolée, consistant en une protéine DNase I humaine, recombinante et exprimée par une plante présentant un résidu de glycine comme acide aminé N-terminal, ladite protéine DNase I humaine, recombinante présentant une susceptibilité réduite à une inhibition d'actine de l'activité d'endonucléase par comparaison avec celle d'une DNase I humaine, recombinante et exprimée par une cellule de mammifère, dans laquelle ladite protéine DNase I humaine, recombinante et exprimée par une plante présente la séquence d'acides aminés telle que spécifiée dans SEQ ID NO : 5, et dans laquelle ladite protéine DNase I humaine, recombinante et exprimée par une plante présente au moins un core α-(1,3) fucose.
 
2. Cellule végétale transformée de manière stable ou transitoire exprimant par voie recombinante la protéine DNase I humaine selon la revendication 1.
 
3. Procédé de production de la protéine DNase I humaine, recombinante selon la revendication 1, comprenant les étapes consistant à :

fournir la cellule végétale transformée de manière stable ou transitoire selon la revendication 2, et

cultiver ladite cellule végétale de manière à produire ladite protéine DNase I humaine, recombinante et comprenant, en outre et facultativement, l'étape pour isoler ladite protéine DNase I humaine, recombinante à partir de ladite cellule végétale.


 
4. Protéine DNase humaine selon la revendication 1, présentant une constante de Michaelis inférieure (Km) et une vélocité de réaction supérieure (Vmax) pour l'activité d'endonucléase par comparaison avec celle de la DNase I humaine, recombinante et exprimée par une cellule de mammifère.
 
5. Composition pharmaceutique comprenant, comme ingrédient actif, la protéine DNase I humaine isolée selon l'une quelconque des revendications 1 à 4, et un vecteur acceptable sur le plan pharmaceutique.
 
6. Composition pharmaceutique selon la revendication 5, dans laquelle ladite protéine DNase I humaine isolée est une protéine DNase I humaine pure à au moins 90-95%, ou dans laquelle la composition pharmaceutique comprend en outre une protéine enzymatique de plante bêta-acétylhexosaminidase.
 
7. Composition pharmaceutique selon la revendication 5 destinée à être utilisée pour traiter une maladie liée à la DNase I ou un état dans un sujet ayant besoin de celle-ci.
 
8. Composition pharmaceutique selon la revendication 7 destinée à être utilisée pour traiter une maladie liée à la DNase I ou un état dans un sujet ayant besoin de celle-ci, en réduisant le module visqueux d' expectoration.
 
9. Composition pharmaceutique selon la revendication 7 destinée à être utilisée pour traiter une maladie liée à la DNase I ou un état dans un sujet ayant besoin de celle-ci, en réduisant le module élastique d' expectoration.
 
10. Composition pharmaceutique selon la revendication 5 destinée à être utilisée pour traiter une fibrose kystique (FK) dans un sujet ayant besoin de celle-ci.
 
11. Composition pharmaceutique selon la revendication 5 destinée à être utilisée pour traiter une bronchopneumopathie chronique obstructive (BPCO) dans un sujet ayant besoin de celle-ci.
 




Drawing



























































Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description