U.S. patent application number 10/990207 was filed with the patent office on 2005-06-16 for human dnase ii.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Baker, Kevin P., Baron, Will F..
Application Number | 20050130209 10/990207 |
Document ID | / |
Family ID | 24563530 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130209 |
Kind Code |
A1 |
Baker, Kevin P. ; et
al. |
June 16, 2005 |
Human DNase II
Abstract
This invention relates to a novel human deoxyribonuclease,
referred to as human DNase II. The invention provides nucleic acid
sequences encoding human DNase II, thereby enabling the production
of human DNase II by recombinant DNA methods in quantities
sufficient for clinical use. The invention also relates to
pharmaceutical compositions and diagnostic and therapeutic uses of
human DNase II.
Inventors: |
Baker, Kevin P.;
(Darnestown, MD) ; Baron, Will F.; (Moorpark,
CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
24563530 |
Appl. No.: |
10/990207 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10990207 |
Nov 15, 2004 |
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10408167 |
Apr 4, 2003 |
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10408167 |
Apr 4, 2003 |
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09861034 |
May 18, 2001 |
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6569429 |
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09861034 |
May 18, 2001 |
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08639294 |
Apr 25, 1996 |
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6265195 |
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Current U.S.
Class: |
435/6.12 ;
435/199; 435/320.1; 435/325; 435/6.13; 435/69.1; 530/388.26;
536/23.2 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 31/10 20180101; A61P 9/00 20180101; A61P 37/02 20180101; C12N
9/22 20130101; A61P 19/02 20180101; A61P 43/00 20180101; A61P 31/00
20180101; A61P 1/02 20180101; A61P 1/00 20180101; A61K 38/00
20130101; A61P 17/00 20180101; A61P 11/06 20180101; A61P 37/00
20180101; A61P 1/18 20180101; A61P 1/16 20180101; A61P 13/12
20180101; A61P 37/06 20180101; A61P 11/02 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/320.1; 435/325; 536/023.2; 530/388.26 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/16; C12N 009/22 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding the amino acid sequence shown in FIG. 1 for
mature human DNase II.
2. An expression vector comprising a nucleotide sequence of claim 1
encoding the amino acid sequence shown in FIG. 1 for mature human
DNase II operably linked to a promoter recognized by a host cell
transformed with the vector.
3. The isolated nucleic acid molecule of claim 1 comprising a
nucleotide sequence that encodes an amino acid sequence having at
least 95% identity with the amino acid sequence shown in FIG. 1 for
mature human DNase II.
4. The isolated nucleic acid molecule of claim 3 comprising a
nucleotide sequence that encodes an amino acid sequence that
differs from the amino acid sequence shown in FIG. 1 for mature
human DNase II by the substitution of one amino acid for another at
only a single position within the FIG. 1 sequence.
5. The isolated nucleic acid molecule of claim 3 comprising a
nucleotide sequence that encodes an amino acid sequence that
differs from the amino acid sequence shown in FIG. 1 for mature
human DNase II by the substitution of one amino acid for another at
only two positions within the FIG. 1 sequence.
6. A host cell transformed with an expression vector of claim 2
comprising a nucleotide sequence encoding the amino acid sequence
shown in FIG. 1 for mature human DNase II.
7. A method for producing human DNase II comprising: (a)
transforming a cell containing an endogenous human DNase II gene
with a homologous DNA comprising an amplifiable gene and a flanking
sequence of at least about 150 base pairs that is homologous with a
DNA sequence within or in proximity to the DNase II gene, whereby
the homologous DNA integrates into the cell genome by
recombination; (b) culturing the cells under conditions that select
for amplification of the amplifiable gene, whereby the human DNase
II gene is also amplified; and thereafter (c) recovering human
DNase II from the cells.
8. An isolated polypeptide comprising the amino acid sequence shown
in FIG. 1 for mature human DNase II.
9. An isolated polypeptide comprising an amino acid sequence having
at least 95% identity with the amino acid sequence shown in FIG. 1
for mature human DNase II, which polypeptide has DNA-hydrolytic
activity.
10. The isolated polypeptide of claim 9 comprising an amino acid
sequence that differs from the amino acid sequence shown in FIG. 1
for mature human DNase II by the substitution of one amino acid for
another at only a single position within the FIG. 1 sequence.
11. A polypeptide of claim 12 wherein the amino acid substitution
creates a glycosylation site within the polypeptide that is not
present in naturally-occurring human DNase II.
12. An antibody that is capable of binding to the amino acid
sequence shown in FIG. 1 for mature human DNase II.
13. The antibody of claim 16 that is a monoclonal antibody.
14. A method for the treatment of a patient having a pulmonary
disease or disorder comprising administering to the patient a
therapeutically effective amount of human DNase II according to
claim 9.
15. The method of claim 18 wherein the disease or disorder is
cystic fibrosis.
16. A method for the treatment of a patient having systemic lupus
erythematosus comprising administering to the patient a
therapeutically effective amount of human DNase II according to
claim 9.
Description
RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 09/861,034,
filed May 18, 2001, claiming priority to application U.S. Ser. No.
08/639,294, filed Apr. 25, 1996, now U.S. Pat. No. 6,265,195 B1,
the entire disclosures of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to newly identified human
deoxyribonuclease (DNase) protein, nucleic acid encoding such
protein, the use of such protein and nucleic acid, as well as the
production of such protein and nucleic acid, for example, by
recombinant DNA methods.
BACKGROUND OF THE INVENTION
[0003] Deoxyribonuclease (DNase) is a phosphodiesterase capable of
hydrolyzing polydeoxyribonucleic acid, and is known to occur in
several molecular forms. Based on their biochemical properties and
enzymatic activities, DNase proteins have been classified as two
types, DNase I and DNase II. DNase I proteins have a pH optimum
near neutrality, an obligatory requirement for divalent cations,
and produce 5'-phosphate nucleotides on hydrolysis of DNA. DNase II
proteins exhibit an acid pH optimum, do not require divalent
cations for activity, and produce 3'-phosphate nucleotides on
hydrolysis of DNA.
[0004] DNase from various species have been purified to a varying
degree. For example, various forms of bovine DNase I have been
purified and completely sequenced (Liao, et al., J. Biol. Chem.
248:1489-1495 (1973); Oefner, et al., J. Mol. Biol. 192:605-632
(1986); Lahm, et al., J. Mol. Biol. 221:645-667 (1991)), and DNA
encoding bovine DNase I has been cloned and expressed (Worrall, et
al., J. Biol. Chem 265:21889-21895 (1990)). Porcine and orcine
DNase I proteins also have been purified and completely sequenced
(Paudel, et al., J. Biol. Chem. 261:16006-16011 (1986); Paudel, et
al., J. Biol. Chem. 261 :16012-16017 (1986)).
[0005] DNA encoding a human DNase I has been isolated and sequenced
and the DNA has been expressed in recombinant host cells, thereby
enabling the production of human DNase I in commercially useful
quantities. Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192
(1990). The term "human DNase I" will be used hereafter to refer to
the mature polypeptide disclosed in Shak, et al.
[0006] DNA encoding other polypeptides having homology to human
DNase I also have been identified. Rosen, et al., PCT Patent
Publication No. WO 95/30428, published Nov. 16, 1995; and Parrish,
et al., Hum. Mol. Genet. 4:1557-1564 (1995).
[0007] DNase I has a number of known utilities and has been used
for therapeutic purposes. Its principal therapeutic use 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. See e.g.,
Lourenco, et al., Arch. Intern. Med. 142:2299-2308 (1982); Shak, et
al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990); Hubbard, et al.,
New Engl. J. Med. 326:812-815 (1992); Fuchs, et al., New Engl. J.
Med. 331:637-642 (1994); Bryson, et al., Drugs 48:894-906 (1994).
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, or degrading, high-molecular-weight DNA that is
present in such secretions. Shak, et al., Proc. Natl. Acad. Sci.
87:9188-9192 (1990); Aitken, et al., J. Am. Med. Assoc.
267:1947-1951 (1992).
[0008] Various forms of DNase II also have reportedly been
purified, including bovine DNase II (Lesca, J. Biol. Chem.
251:116-123 (1976)), human DNase II (Yamanaku, et al., J. Biol.
Chem. 249:3884-3889 (1974); Murai, et al., J. Biochem. 87:1097-1103
(1980); Harosh, et al., Eur. J. Biochem. 202:479-484 (1991);
Yasuda, et al., Biochem. Biophys. Acta 1119:185-193 (1992)),
porcine DNase II (Bernardi, et al., Biochemistry 4:1725-1729
(1965); Liao, et al., J. Biol. Chem. 260:10708-10713 (1990)), and
rat DNase II (Dulaney, et al., J. Biol. Chem. 247:1424-1432
(1972)). The physical properties of the human DNase II proteins
described in these reports vary considerably (e.g., reported
molecular weights range from 32,000 to 45,000 Daltons), which leads
to uncertainty whether there is one or multiple naturally occurring
forms of the human protein.
[0009] Recent interest in human DNase II has arisen because of its
possible role in the programmed cell death process of apoptosis
(Barry, et al., Arch. Biochem. Biophys. 300:440-450 (1993); Barry,
et al., Cancer Res. 53:2349-2357 (1993)). One of the events that is
characteristic of that process is the degradation of nuclear DNA
into nucleosomal fragments. The ability to prevent or inhibit the
expression of human DNase II or its enzymatic activity within human
cells may be important in preventing or limiting such intracellular
destruction of DNA, and thus may be an effective means of
interrupting the process of apoptosis. In other instances, it may
be useful to increase the expression of human DNase II within a
certain population of cells within a human patient, such as cancer
cells, in order to induce apoptosis of those cells.
SUMMARY OF THE INVENTION
[0010] The present invention provides human DNase II protein, as
well as analogs and variants thereof, that have DNA-hydrolytic
activity. As is characteristic of DNase II proteins in general, the
human DNase II of the present invention exhibits an acid pH
optimum, and does not require divalent cation for activity.
[0011] The invention also provides nucleic acids encoding human
DNase II, recombinant vectors comprising such nucleic acids,
recombinant host cells transformed with those nucleic acids or
vectors, and processes for producing human DNase II by means of
recombinant DNA technology. The invention includes the use of such
nucleic acids and vectors for in vivo or ex vivo gene therapy.
[0012] The invention also provides complementary nucleic acids,
including so-called anti-sense oligonucleotides, that are capable
of binding to and preventing the expression of nucleic acid within
a cell that encodes human DNase II.
[0013] The invention also provides pharmaceutical compositions
comprising human DNase II, optionally together with a
pharmaceutically acceptable excipient, as well as substantially
purified antibodies that are capable of binding to human DNase
II.
[0014] The invention also provides methods for reducing the
viscoelasticity or viscous consistency of DNA-containing material
in a patient, comprising administering a therapeutically effective
dose of human DNase II to the patient. The invention is
particularly directed to a method of treating a patient having a
disease such as cystic fibrosis, chronic bronchitis, pneumonia,
bronchiectasis, emphysema, asthma, or systemic lupus erythematosus,
that comprises administering a therapeutically effective amount of
human DNase II to the patient. The invention also is directed to
the use of human DNase II in vitro, such as for degrading DNA that
is present in a biological specimen or other material, and in
diagnostic and other assays.
[0015] These and other aspects of the invention will be apparent to
the ordinary skilled artisan upon consideration of the following
detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A-1B show the nucleotide sequence (SEQ. ID. NO: 1) and
deduced amino acid sequence (SEQ. ID. NO: 2) of human DNase II. The
predicted leader (signal amino acid sequence is underlined and the
start of the mature protein is indicated by the arrowhead. The
eight cysteine residues are indicated by asterisks and potential
N-linked glycosylation sites are boxed.
DETAILED DESCRIPTION
[0017] The various aspects of the present invention are
accomplished by first providing isolated DNA comprising the
nucleotide coding sequence for human DNase II. By providing the
full nucleotide coding sequence for human DNase II, the invention
enables the production of human DNase II by means of recombinant
DNA technology, thereby making available for the first time
sufficient quantities of substantially pure human DNase II protein
for diagnostic and therapeutic uses.
[0018] As used herein, the term "human DNase II" refers to the
polypeptide having the amino acid sequence of the mature protein
set forth in FIG. 1 as well as modified and variant forms thereof
as described herein. Modified and variant forms of human DNase II
are produced in vitro by means of chemical or enzymatic treatment
or in vivo by means of recombinant DNA technology. Such
polypeptides differ from human DNase II, for example, by virtue of
one or more amino acid substitutions, insertions, and/or deletions,
or in the extent or pattern of glycosylation, but in all cases will
possess DNA-hydrolytic activity. A "variant" or "amino acid
sequence variant" of human DNase II is a polypeptide that comprises
an amino acid sequence different from that of human DNase II.
Generally, a variant will have at least 80% sequence identity,
preferably at least 90% sequence identity, more preferably at least
95% sequence identity, and most preferably at least 98% sequence
identity with human DNase II. Percentage sequence identity is
determined, for example, by the Fitch, et al., Proc. Natl. Acad.
Sci. USA 80:1382-1386 (1983), version of the algorithm described by
Needleman, et al., J. Mol. Biol. 48:443-453 (1970), after aligning
the sequences to provide for maximum homology. Such variants
include naturally occurring allelic forms of human DNase II that
are of human origin as well as naturally occurring homologs of
human DNase II that are found in other animal species.
[0019] "DNA-hydrolytic activity" refers to the enzymatic activity
of human DNase II in hydrolyzing (cleaving) substrate DNA to yield
3'-phosphorylated oligonucleotide end products. DNA-hydrolytic
activity is readily determined by any of several different methods
known in the art, including analytical polyacrylamide and agarose
gel electrophoresis, hyperchromicity assay (Kunitz, J. Gen.
Physiol. 33:349-362 (1950); Kunitz, J. Gen. Physiol. 33:363-377
(1950)), or methyl green assay (Kurnick, Arch. Biochem. 29:41-53
(1950); Sinicropi, et al., Anal. Biochem. 222:351-358 (1994)). As a
routine matter, the pH and buffer used in these methods are varied
so as to provide the conditions wherein the particular human DNase
II will exhibit such activity, if any.
[0020] For convenience, substitutions, insertions, and/or deletions
in the amino acid sequence of human DNase II are usually made by
introducing mutations into the corresponding nucleotide sequence of
the DNA encoding human DNase II, for example by site-directed
mutagenesis. Expression of the mutated DNA then results in
production of the variant human DNase II, having the desired amino
acid sequence.
[0021] Whereas any technique known in the art can be used to
perform site-directed mutagenesis, e.g. as disclosed in Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second Edition
(Cold Spring Harbor Laboratory Press, New York (1989)),
oligonucleotide-directed mutagenesis is the preferred method for
preparing the human DNase II variants of this invention. This
method, which is well known in the art (Zoller, et al., Meth.
Enzymol. 100:4668-500 (1983); Zoller, et al., Meth. Enzymol.
154:329-350 (1987); Carter, Meth. Enzymol. 154:382-403 (1987);
Kunkel, et al., Meth. Enzymol. 154:367-382 (1987); Horwitz, et al.,
Meth. Enzymol. 185:599-611 (1990)), is particularly suitable for
making substitution variants, although it may also be used to
conveniently prepare deletion and insertion variants, as well as
variants having multiple substitution, insertion, and/or deletion
mutations.
[0022] Briefly, in carrying out site-directed mutagenesis of DNA
encoding human DNase II (or a variant thereof), the DNA is altered
by first hybridizing an oligonucleotide encoding the desired
mutation to a single strand of the DNA. After hybridization, a DNA
polymerase is used to synthesize an entire second strand, using the
hybridized oligonucleotide as a primer, and using the single strand
of the DNA as a template. Thus, the oligonucleotide encoding the
desired mutation is incorporated in the resulting double-stranded
DNA.
[0023] Oligonucleotides may be prepared by any suitable method,
such as by purification of a naturally occurring DNA or by in vitro
synthesis. For example, oligonucleotides are readily synthesized
using various techniques in organic chemistry, such as described by
Narang, et al., Meth. Enzymol. 68:90-98 (1979); Brown, et al.,
Meth. Enzymol. 68:109-151 (1979); Caruthers, et al., Meth. Enzymol.
154:287-313 (1985). The general approach to selecting a suitable
oligonucleotide for use in site-directed mutagenesis is well known.
Typically, the oligonucleotide will contain 10-25 or more
nucleotides, and will include at least 5 nucleotides on either side
of the sequence encoding the desired mutation so as to ensure that
the oligonucleotide will hybridize preferentially at the desired
location to the single-stranded DNA template molecule.
[0024] "Polymerase chain reaction," or "PCR," generally refers to a
method for amplification of a desired nucleotide sequence in vitro,
as described, for example, in U.S. Pat. No. 4,683,195. In general,
the PCR method involves repeated cycles of primer extension
synthesis, using oligonucleotide primers capable of hybridizing
preferentially to a template nucleic acid.
[0025] PCR mutagenesis (Higuchi, in PCR Protocols, pp.177-183
(Academic Press, 1990); Vallette, et al., Nuc. Acids Res.
17:723-733 (1989)) is also suitable for making the variants of
human DNase II. Briefly, when small amounts of template DNA are
used as starting material in a PCR, primers that differ slightly in
sequence from the corresponding region in the template DNA can be
used to generate relatively large quantities of a specific DNA
fragment that differs from the template sequence only at the
positions where the primers differ from the template. For
introduction of a mutation into a plasmid DNA, for example, the
sequence of one of the primers includes the desired mutation and is
designed to hybridize to one strand of the plasmid DNA at the
position of the mutation; the sequence of the other primer must be
identical to a nucleotide sequence within the opposite strand of
the plasmid DNA, but this sequence can be located anywhere along
the plasmid DNA. It is preferred, however, that the sequence of the
second primer is located within 200 nucleotides from that of the
first, such that in the end the entire amplified region of DNA
bounded by the primers can be easily sequenced. PCR amplification
using a primer pair like the one just described results in a
population of DNA fragments that differ at the position of the
mutation specified by the primer, and possibly at other positions,
as template copying is somewhat error-prone. Wagner, et al., in PCR
Topics, pp.69-71 (Springer-Verlag, 1991).
[0026] If the ratio of template to product amplified DNA is
extremely low, the majority of product DNA fragments incorporate
the desired mutation(s). This product DNA is used to replace the
corresponding region in the plasmid that served as PCR template
using standard recombinant DNA methods. Mutations at separate
positions can be introduced simultaneously by either using a mutant
second primer, or performing a second PCR with different mutant
primers and ligating the two resulting PCR fragments simultaneously
to the plasmid fragment in a three (or more)-part ligation.
[0027] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al., Gene,
34:315-323 (1985). The starting material is the plasmid (or other
vector) comprising the DNA sequence to be mutated. The codon(s) in
the starting DNA to be mutated are identified. There must be a
unique restriction endonuclease site on each side of the identified
mutation site(s). If no such restriction sites exist, they may be
generated using the above-described oligonucleotide-mediated
mutagenesis method to introduce them at appropriate locations in
the DNA. The plasmid DNA is cut at these sites to linearize it. A
double-stranded oligonucleotide encoding the sequence of the DNA
between the restriction sites but containing the desired
mutation(s) is synthesized using standard procedures, wherein the
two strands of the oligonucleotide are synthesized separately and
then hybridized together using standard techniques. This
double-stranded oligonucleotide is referred to as the cassette.
This cassette is designed to have 5' and 3' ends that are
compatible with the ends of the linearized plasmid, such that it
can be directly ligated to the plasmid. The resulting plasmid
contains the mutated DNA sequence.
[0028] The presence of mutation(s) in a DNA is determined by
methods well known in the art, including restriction mapping and/or
DNA sequencing. A preferred method for DNA sequencing is the
dideoxy chain termination method of Sanger, et al., Proc. Natl.
Acad. Sci. USA 72:3918-3921 (1979).
[0029] DNA encoding human DNase II is inserted into a replicable
vector for further cloning or expression. "Vectors" are plasmids
and other DNAs that are capable of replicating within a host cell,
and as such, are useful for performing two functions in conjunction
with compatible host cells (a vector-host system). One function is
to facilitate the cloning of nucleic acid that encodes human DNase
II, i.e., to produce usable quantities of the nucleic acid. The
other function is to direct the expression of human DNase II. One
or both of these functions are performed by the vector in the
particular host cell used for cloning or expression. The vectors
will contain different components depending upon the function they
are to perform.
[0030] The human DNase II of the present invention may be expressed
in the form of a preprotein wherein the DNase II includes a leader
or signal sequence, or may be in the form of a mature protein which
lacks a leader or signal sequence. The human DNase II also may be
in the form of a fusion protein wherein additional amino acid
residues are covalently joined to the amino- or carboxy-terminus of
the preprotein or mature form of the DNase.
[0031] To produce human DNase II, an expression vector will
comprise DNA encoding human DNase II, as described above, operably
linked to a promoter and a ribosome binding site. The human DNase
II then is expressed directly in recombinant cell culture, or as a
fusion with a heterologous polypeptide, preferably a signal
sequence or other polypeptide having a specific cleavage site at
the junction between the heterologous polypeptide and the human
DNase II amino acid sequence.
[0032] "Operably linked" refers to the covalent joining of two or
more DNA sequences, by means of enzymatic ligation or otherwise, in
a configuration relative to one another such that the normal
function of the sequences can be performed. For example, DNA for a
presequence or secretory leader is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, then synthetic oligonucleotide adaptors or
linkers are used, in conjunction with standard recombinant DNA
methods.
[0033] Prokaryotes (e.g., E. coli, strains of Bacillus,
Pseudomonas, and other bacteria) are the preferred host cells for
the initial cloning steps of this invention. They are particularly
useful for rapid production of large amounts of DNA, for production
of single-stranded DNA templates used for site-directed
mutagenesis, and for DNA sequencing of the variants generated.
Prokaryotic host cells also may be used for expression of DNA
encoding human DNase II. Polypeptides that are produced in
prokaryotic cells typically will be non-glycosylated.
[0034] In addition, human DNase II may be expressed in eukaryotic
host cells, including eukaryotic microbes (e.g., yeast) or cells
derived from an animal or other multicellular organism (e.g.,
Chinese hamster ovary cells, and other mammalian cells), or in live
animals (e.g., cows, goats, sheep). Insect cells and fungii also
may be used.
[0035] Cloning and expression methodologies are well known in the
art. Examples of prokaryotic and eukaryotic host cells, and
starting expression vectors, suitable for use in producing human
DNase II are, for example, those disclosed in Shak, PCT Patent
Publication No. WO 90/07572, published Jul. 12, 1990. To obtain
expression of human DNase II, an expression vector of the invention
is introduced into host cells by transformation or transfection,
and the resulting recombinant host cells are cultured in
conventional nutrient media, modified as appropriate for inducing
promoters, selecting recombinant cells, or amplifying human DNase
II DNA. The culture conditions, such as temperature, pH, and the
like, are those previously used with the host cell, and as such
will be apparent to the ordinarily skilled artisan.
[0036] "Transformation" and "transfection" are used interchangeably
to refer to the process of introducing DNA into a cell. Following
transformation or transfection, the DNA may integrate into the host
cell genome, or may exist as an extrachromosomal element. If
prokaryotic cells or cells that contain substantial cell wall
constructions are used as hosts, the preferred methods of
transfection of the cells with DNA is the calcium treatment method
described by Cohen et al., Proc. Natl. Acad. Sci. 69:2110-2114
(1972) or the polyethylene glycol method of Chung et al., Nuc.
Acids. Res. 16:3580 (1988). If yeast are used as the host,
transfection is generally accomplished using polyethylene glycol,
as taught by Hinnen, Proc. Natl. Acad. Sci. U.S.A., 75: 1929-1933
(1978). If mammalian cells are used as host cells, transfection
generally is carried out by the calcium phosphate precipitation
method, Graham, et al., Virology 52:546 (1978), Gorman, et al., DNA
and Protein Eng. Tech. 2:3-10 (1990). However, other known methods
for introducing DNA into prokaryotic and eukaryotic cells, such as
nuclear injection, electroporation, or protoplast fusion also are
suitable for use in this invention.
[0037] Particularly useful in this invention are expression vectors
that provide for the transient expression in mammalian cells of DNA
encoding human DNase II. In general, transient expression involves
the use of an expression vector that is able to efficiently
replicate in a host cell, such that the host cell accumulates many
copies of the expression vector and, in turn, synthesizes high
levels of a desired polypeptide encoded by the expression vector.
Transient expression systems, comprising a suitable expression
vector and a host cell, allow for the convenient positive
identification of polypeptides encoded by cloned DNAs, as well as
for the rapid screening of such polypeptides for desired biological
or physiological properties. Wong, et al., Science 228:810-815
(1985); Lee, et al., Proc. Nat Acad. Sci. USA 82:4360-4364 (1985);
Yang, et al., Cell 47:3-10 (1986). Thus, transient expression
systems are conveniently used for expressing the DNA encoding amino
acid sequence variants of human DNase II, in conjunction with
assays to identify those variants that have such useful properties
as increased half-life or decreased immunogenicity in vivo, or
increased DNA hydrolytic activity at physiological pH.
[0038] Human DNase II preferably is secreted from the host cell in
which it is expressed, in which case the variant is recovered from
the culture medium in which the host cells are grown. In that case,
it may be desirable to grow the cells in a serum free culture
medium, since the absence of serum proteins and other serum
components in the medium may facilitate purification of the
variant. If it is not secreted, then the human DNase II is
recovered from lysates of the host cells. When the human DNase II
is expressed in a host cell other than one of human origin, the
variant will be completely free of proteins of human origin. In any
event, it will be necessary to purify the human DNase II from
recombinant cell proteins in order to obtain substantially
homogeneous preparations of the human DNase II. For therapeutic
uses, the purified human DNase II preferably will be greater than
99% pure (i.e., any other proteins will comprise less than 1% of
the total protein in the purified composition).
[0039] It is further contemplated that human DNase II may be
produced by a method involving homologous recombination and
amplification, for example, as described in PCT Patent Publication
No. WO 91/06667, published May 16, 1991. Briefly, this method
involves transforming cells containing an endogenous gene encoding
human DNase II with a homologous DNA, which homologous DNA
comprises (1) an amplifiable gene (e.g., a gene encoding
dihydrofolate reductase (DHFR)), and (2) at least one flanking
sequence, having a length of at least about 150 base pairs, which
is homologous with a nucleotide sequence in the cell genome that is
within or in proximity to the gene encoding human DNase II. The
transformation is carried out under conditions such that the
homologous DNA integrates into the cell genome by recombination.
Cells having integrated the homologous DNA then are subjected to
conditions which select for amplification of the amplifiable gene,
whereby the human DNase II gene amplified concomitantly. The
resulting cells then are screened for production of desired amounts
of human DNase II. Flanking sequences that are in proximity to a
gene encoding human DNase II are readily identified, for example,
by the method of genomic walking, using as a starting point the
nucleotide sequence of human DNase II shown in FIG. 1. Spoerel, et
al., Meth. Enzymol. 152:598-603 (1987).
[0040] Generally, purification of human DNase II is accomplished by
taking advantage of the differential physicochemical properties of
the human DNase II as compared to the contaminants with which it
may be associated. For example, as a first step, the culture medium
or host cell lysate is centrifuged to remove particulate cell
debris. The human DNase II thereafter is purified from contaminant
soluble proteins and polypeptides, for example, by ammonium sulfate
or ethanol precipitation, gel filtration (molecular exclusion
chromatography), ion-exchange chromatography, hydrophobic
chromatography, immunoaffinity chromatography (e.g., using a column
comprising anti-human DNase II antibodies coupled to Sepharose),
tentacle cation exchange chromatography (Frenz, et al., U.S. Pat.
No. 5,279,823, issued Jan. 18, 1994), reverse phase HPLC, and/or
gel electrophoresis.
[0041] In some host cells (especially bacterial host cells) the
human DNase II may be expressed initially in an insoluble,
aggregated form (referred to in the art as "refractile bodies" or
"inclusion bodies") in which case it will be necessary to
solubilize and renature the human DNase II in the course of its
purification. Methods for solubilizing and renaturing recombinant
protein refractile bodies are known in the art (see e.g., Builder,
et al., U.S. Pat. No. 4,511,502, issued Apr. 16, 1985).
[0042] In another embodiment of this invention, covalent
modifications are made directly to human DNase II to give it a
desired property (for example, increased half-life or decreased
immunogenicity in vivo, or increased DNA hydrolytic activity at
physiological pH), and may be made instead of or in addition to the
amino acid sequence substitution, insertion, and deletion mutations
described above.
[0043] Covalent modifications are introduced by reacting targeted
amino acid residues of human DNase II with an organic derivatizing
agent that is capable of reacting with selected amino acid
side-chains or N- or C-terminal residues. Suitable derivatizing
agents and methods are well known in the art. Covalent coupling of
glycosides to amino acid residues of the protein may be used to
modify or increase the number or profile of carbohydrate
substituents.
[0044] The covalent attachment of agents such as polyethylene
glycol (PEG) or human serum albumin to human DNase II may reduce
immunogenicity and/or toxicity of the human DNase II and/or prolong
its half-life, as has been observed with other proteins.
Abuchowski, et al., J. Biol. Chem. 252:3582-3586 (1977); Poznansky,
et al., FEBS Letters 239:18-22 (1988); Goodson, et al.,
Biotechnology 8:343-346 (1990); Katre, J. Immunol. 144:209-213
(1990); Harris, Polyethylene Glycol Chemistry (Plenum Press, 1992).
As another example, the variant or modified form of human DNase II
may comprise an amino acid sequence mutation or other covalent
modification that reduces the susceptibility of the variant to
degradation by proteases (e.g., neutrophil elastase) that may be
present in sputum and other biological materials, as compared to
human DNase II.
[0045] Antibodies to human DNase II are produced by immunizing an
animal with human DNase II or a fragment thereof, optionally in
conjunction with an immunogenic polypeptide, and thereafter
recovering antibodies from the serum of the immunized animals.
Alternatively, monoclonal antibodies are prepared from cells of the
immunized animal in conventional fashion. The antibodies also can
be made in the form of chimeric (e.g., humanized) or single chain
antibodies or Fab fragments, using methods well known in the art.
Preferably, the antibodies will bind to human DNase II but will not
substantially bind to (i.e., cross react with) other DNase proteins
(such as human and bovine DNase I). The antibodies can be used in
methods relating to the localization and activity of human DNase
II, for example, for detecting human DNase II and measuring its
levels in tissues or clinical samples. Immobilized anti-human DNase
II antibodies are particularly useful in the detection of human
DNase II in clinical samples for diagnostic purposes, and in the
purification of human DNase II.
[0046] Purified human DNase II is used to reduce the
viscoelasticity of DNA-containing material, such as sputum, mucus,
or other pulmonary secretions. human DNase II is particularly
useful for the treatment of patients with pulmonary disease who
have abnormal viscous or inspissated secretions and conditions such
as acute or chronic bronchial pulmonary disease, including
infectious pneumonia, bronchitis or tracheobronchitis,
bronchiectasis, cystic fibrosis, asthma, tuberculosis, and fungal
infections. For such therapies, a solution or finely divided dry
preparation of the human DNase II is instilled in conventional
fashion into the airways (e.g., bronchi) or lungs of a patient, for
example by aerosolization.
[0047] Human DNase II also is useful for adjunctive treatment of
abscesses or severe closed-space infections in conditions such as
empyema, meningitis, abscess, peritonitis, sinusitis, otitis,
periodontitis, pericarditis, pancreatitis, cholelithiasis,
endocarditis and septic arthritis, as well as in topical treatments
in a variety of inflammatory and infected lesions such as infected
lesions of the skin and/or mucosal membranes, surgical wounds,
ulcerative lesions and burns. Human DNase II may improve the
efficacy of antibiotics used in the treatment of such infections
(e.g., gentamicin activity is markedly reduced by reversible
binding to intact DNA).
[0048] Human DNase II also is useful for preventing the new
development and/or exacerbation of respiratory infections, such as
may occur in patients having cystic fibrosis, chronic bronchitis,
asthma, pneumonia, or other pulmonary disease, or patients whose
breathing is assisted by ventilator or other mechanical device, or
other patients at risk of developing respiratory infections, for
example post-surgical patients.
[0049] Human DNase II also is useful for the treatment for systemic
lupus erythematosus (SLE), a life-threatening autoimmune disease
characterized by the production of diverse autoantibodies. DNA is a
major antigenic component of the immune complexes. In this
instance, the human DNase II may be given systemically, for example
by intravenous, subcutaneous, intrathecal, or intramuscular
administration to the affected patient.
[0050] Finally, human DNase II is useful for the treatment of other
non-infected conditions in which there is an accumulation of
cellular debris that includes cellular DNA, such as pyelonephritis
and tubulo-interstitial kidney disease.
[0051] Human DNase II can be formulated according to known methods
to prepare therapeutically useful compositions. Typically, the
human DNase II is formulated with a physiologically acceptable
excipient (or carrier) for therapeutic use. Such excipients are
used, for example, to provide liquid formulations and
sustained-release formulations of human DNase II. The human DNase
II formulation may be used with commercially-available nebulizers
including jet nebulizers and ultrasonic nebulizers for
administration of the DNase II directly into the airways or lungs
of an affected patient. Another preferred therapeutic composition
is a dry powder of human DNase II, preferably prepared by
spray-drying of a solution of the human DNase II. In all cases, it
is desirable that the therapeutic compositions of DNase II be
sterile. Preferably, the therapeutic compositions are disposed in a
container fabricated of plastic or other non-glass material.
[0052] In a further embodiment, the therapeutic composition
comprises cells actively producing human DNase II. Such cells may
be directly introduced into the tissue of a patient, or may be
encapsulated within porous membranes which are then implanted in a
patient (see e.g., Aebischer, et al., U.S. Pat. No. 4,892,538,
issued Jan. 9, 1990; Aebischer, et al., U.S. Pat. No. 5,283,187,
issued Feb. 1, 1994), in either case providing for the delivery of
the human DNase II into areas within the body of the patient in
need of increased concentrations of DNA-hydrolytic activity. In one
embodiment of the invention, the patient's cells are transformed,
either in vivo or ex vivo, with DNA encoding human DNase II, and
then used to produce the human DNase II directly within the
patient. This latter method is commonly referred to as gene
therapy. In another embodiment, the patient's cells are transformed
with other DNA (such as a promoter, enhancer, or amplifiable gene)
that is capable of activating or increasing expression of an
endogenous human DNase II gene.
[0053] In certain circumstances, it may be desirable to decrease
the amount of human DNase II expressed in a cell. For that purpose,
human DNase II anti-sense oligonucleotides can be made and a method
utilized for diminishing the level of human DNase II within the
cell comprising introducing into the cell one or more human DNase
II anti-sense oligonucleotides. The term "human DNase II anti-sense
oligonucleotide" refers to an oligonucleotide that has a nucleotide
sequence that is capable of interacting through base pairing with a
complementary nucleotide sequence that is involved in the
expression of human DNase II within a cell, and thereby interfering
with such expression.
[0054] The therapeutically effective amount of human DNase II will
depend, for example, upon the amount of DNA in the material to be
treated, the therapeutic objectives, the route of administration,
and the condition of the patient. Accordingly, it will be necessary
for the therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. Generally, the therapeutically effective amount of human
DNase II will be a dosage of from about 0.1 .mu.g to about 5 mg of
the variant per kilogram of body weight of the patient,
administered within pharmaceutical compositions, as described
herein.
[0055] Human DNase II optionally is combined with or administered
in concert with 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, published Oct. 13,
1994; 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)).
[0056] This invention also provides methods for determining the
presence of a nucleic acid molecule encoding human DNase II in test
samples prepared from cells, tissues, or biological fluids,
comprising contacting the test sample with isolated DNA comprising
all or a portion of the nucleotide coding sequence for human DNase
II and determining whether the isolated DNA hybridizes to a nucleic
acid molecule in the test sample. DNA comprising all or a portion
of the nucleotide coding sequence for human DNase II is also used
in hybridization assays to identify and to isolate nucleic acids
sharing substantial sequence identity to the coding sequence for
human DNase II, such as nucleic acids that encode
naturally-occurring allelic variants of human DNase II.
[0057] Also provided is a method for amplifying a nucleic acid
molecule encoding human DNase II that is present in a test sample,
comprising the use of an oligonucleotide having a portion of the
nucleotide coding sequence for human DNase II as a primer in a
polymerase chain reaction.
[0058] The following examples are offered by way of illustration
only and are not intended to limit the invention in any manner. All
patent and literature references cited herein are expressly
incorporated.
EXAMPLE 1
Cloning Human DNase II cDNA
[0059] Full-length cDNA encoding human DNase II was identified by
screening a human placental cDNA library (in .lambda.-gt11,
Clontech, Palo Alto, Calif. USA) with a mixture of the following
oligonucleotide probes, each of which had been end-labeled with T4
polynucleotide kinase and .gamma.-.sup.32P-adenosine triphosphate
to a high specific radioactivity:
1 5'-GCCCAGAGAGGGCTGAGTACAAGTATCTGGACGAGAGCTCCGGAGGC-3' (SEQ. ID.
NO: 3) 5'-CCCAGCGCCCCGCAGTCCCAGACACAGATTCCTGGATCTCAGCCC-- 3' (SEQ.
ID. NO: 4) 5'-GAYCARGARGGNGGNTTYTGGCTNAT-3' (SEQ. ID. NO: 5)
5'-GAYCARGARGGNGGNTTYTGGTTRAT-3' (SEQ. ID. NO: 6)
5'-AAYCGNGGNCAYACNAARGGNGT-3' (SEQ. ID. NO: 7)
5'-AAYAGRGGNCAYACNAARGGNGT-3' (SEQ. ID. NO: 8)
[0060] The first two of the oligonucleotide probes listed above
(SEQ. ID. NOS: 3 and 4) comprise portions of the EST sequence
having accession code T53394, in the Genbank database.
[0061] Hybridization of the probes to the cDNA library was carried
out under low stringency conditions (in 20% vol/vol formamide,
5.times.SSPE, 5.times.Denhardt's solution, 0.1% sodium dodecyl
sulfate (SDS), and 100 .mu.g/ml sonicated salmon sperm DNA), at
42.degree. C., for 20 hours. Post hybridization washes were carried
out in 2.times.SSC, 0.1% SDS, at 42.degree. C. 1.times.SSPE is 150
mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4.
1.times.Denhardt's solution is 0.02% Ficoll, 0.02% bovine serum
albumin, and 0.02% polyvinyl-pyrrolidone. 1.times.SSC is 0.15 M
NaCl, 0.015 M sodium citrate, pH 7.0.
[0062] Hybridization-positive phage clones were isolated and their
DNAs sequenced following standard procedures. A 1575 base-pair
insert was identified amongst the hybridization-positive phage
clones, including an open reading frame of 1080 base-pairs that
encodes a predicted protein that is 360 amino acid residues in
length. The nucleotide sequence of the 1575 base-pair insert (SEQ.
ID. NO: 1) and the amino acid sequence of predicted protein (SEQ.
ID. NO: 2) are shown in FIG. 1.
[0063] The predicted protein includes a signal sequence that is 16
amino acid residues in length. Cleavage of the signal sequence
releases the mature protein (human DNase II) that is 344 amino acid
residues in length, and that has a predicted molecular weight of
38,000 Daltons and a predicted pI of 9.0.
EXAMPLE 2
Expression of Human DNase II cDNA
[0064] The cDNA encoding human DNase II was subcloned into a
mammalian expression vector pRK5 (Gorman, et al., DNA and Protein
Engineering Techniques 2:1 (1990); European Patent Publication EP
307,247, published Mar. 15, 1989). The resulting recombinant vector
is referred to as pRK5/human DNase II. Human embryonic kidney 293
cells (American Type Culture Collection, CRL 1573) were grown in
serum-containing Dulbecco Modified Eagle's medium (DMEM) to 70%
confluency and then transiently transfected with pRK5/human DNase
II, or as a control, pRK5 alone. 24 hours post-transfection, the
cells were washed with phosphate buffered saline and transferred to
serum-free medium containing insulin. 72-96 hours later,
conditioned medium was collected from the cell cultures and
concentrated approximately 10-fold. Proteins expressed in the cell
cultures were analyzed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
[0065] Cells transfected with pRK-5/human DNase II were found to
produce a unique protein of about 42,000 -44,000 Daltons, that was
not produced in cells transfected with pRK5 alone.
[0066] The amino-terminal sequence of that secreted protein was
determined by preparing a poly-His tagged version of human DNase
II. DNA encoding the poly-His tagged version of human DNase II was
prepared by joining a nucleotide sequence encoding the amino acid
sequence
[0067] Met-Arg-Gly-Ser-His-His-His-His-His-His (SEQ. ID. NO: 9) to
the 3' end of the nucleotide sequence encoding human DNase II that
is shown in FIG. 1. Human embryonic kidney 293 cells were
transiently transfected with the DNA, and Ni-NTA-silica (Qiagen,
Inc., Chatsworth, California USA) was used to purify the secreted
poly-His tagged human DNase II. The amino-terminal amino acid
sequence of the secreted protein was determined to be
Leu-Thr-Cys-Tyr-Gly-Asp-Ser-Gly-Gln, in agreement with the
predicted amino acid sequence of the mature human DNase II protein
shown in FIG. 1.
EXAMPLE 3
Biological Activity of Human DNase II
[0068] Concentrated cell culture supernatants, prepared as
described above, were tested for DNA-hydrolytic activity in a
hyperchromicity assay (Kunitz, J. Gen. Physiol. 33:349-362 (1950);
Kunitz, J. Gen. Physiol. 33:363-377 (1950)), in which the buffer
used was 0.1M sodium acetate, pH 4.6, 1 mM magnesium chloride. Such
activity was detected in the supernatants from cells transfected
with pRK5/human DNase II, but not in the supernatants from cells
transfected with pRK5 alone. By also assaying cell lysates, it was
determined that approximately 20% -30% of the total human DNase II
activity in the cells transfected with pRK5/human DNase was
secreted.
EXAMPLE 4
Pattern of Expression of Human DNase II in Human Tissue
[0069] Northern blots of various human tissues were performed using
a radiolabeled probe comprising a portion of the coding sequence of
the cloned human DNase II cDNA. Expression of human DNase II
messenger RNA (mRNA) was found in all tissues examined (brain,
colon, heart, small intestine, kidney, liver, lung, peripheral
blood lymphocytes, skeletal muscle, ovary, pancreas, placenta,
prostate, spleen, testis, and thymus)
Sequence CWU 1
1
* * * * *