Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS OF DIAGNOSIS AND TREATMENT OF
INTERSTITIAL LUNG DISEASE
[0001] This application claims the benefit of U.S. Provisional Patent
Application Serial No. 60/431,949, filed December 9, 2002, which
application is hereby incorporated by reference in its entirety.
[0002] This invention was made with government support under Grant Nos.
HL56387 and HL50046 awarded by the National Institutes of Health.
The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] The present invention provides a mammal in which the expression of
one or more lung surfactant protein genes has been suppressed. More
particularly, the invention concerns the inactivating deletion of the
surfactant protein C gene to produce a knockout non-human mammal
with decreased or completely suppressed expression of the endogenous
gene. The invention provides methods for preparing such knockout
mammals and methods of using the knockout mammals to evaluate the
effectiveness of therapeutic agents and regimens to treat diseases or
disorders associated with perturbations in the lung surfactant protein
pathways.
[0004] SP-C is a 34-35 amino acid peptide expressed selectively in type II
epithelial cells in the alveolus of the lung [1,2 for review]. A single SP-
C gene is located on human chromosome 8 that is syntenic to that in
the mouse located on chromosome 14. The SP-C gene encodes a
proprotein of 197 or 191 amino acids (proSP-C) that is palmitoylated,
proteolytically processed and routed through the rough endoplasmic
reticulum and multivesicular bodies to lamellar bodies in which
surfactant is stored. The SP-C peptide is secreted into the airspace
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where it enhances the stability and spreading of phospholipids. The SP-
C peptide is highly hydrophobic and also contains two cysteine
residues in an NH2-terminal domain. These cysteines are palmitoylated
and located near an extended hydrophobic domain wherein 19 of 23
residues are valine, leucine or isoleucine. This hydrophobic region
forms an a-helical structure that spans a lipid bilayer [3]. Both the a
helical domain and the cysteine linked palmitoyl groups are tightly '
associated with phospholipids. SP-C disrupts phospholipid acyl chain
packing and enhances recruitment of phospholipids to monolayers and
multilayers at the air-liquid interface [4,5]. These features suggest a
structural role for SP-C in facilitating the movement of phospholipids
between multilayered films. Biological functions of purified SP-C or
synthetic SP-C peptides are highly active in vitro and in vivo,
enhancing surfactant properties of lipids and restoring lung function in
surfactant deficient animals [6,7]. These results indicate that SP-C
plays an important role in the spreading and stabilization of
phospholipid films in the alveolus.
[0005) An unexpected role for SP-C in pulmonary homeostasis was provided
by recent studies demonstrating that a mutation in the SP-C gene was
associated with idiopathic interstitial pneumonitis (IIP) in humans
[8,9]. Pulmonary disease in these patients was inherited as an
autosomal dominant trait. Interstitial pneumonitis includes various
pulmonary disorders including desquamating interstitial pneumonitis
(D1P), usual interstitial pneumonitis (UIP), nonspecific interstitial
pneumonitis (NSIP), and other disorders broadly termed idiopathic
interstitial pneumonitis (III') [10]. Individuals with these disorders
usually present with progressive lung disease associated with exercise
limitation, tachypnea and shortness of breath. Since mutations in the
SP-C proprotein resulted in the production of an abnormal proSP-C
peptide that was riot fully processed, it has been unclear whether the
lack of SP-C per se or misfolding of proSP-C or SP-C was involved in
the pathogenesis of IIP in these patients [5]. In general, various forms
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of IIP are associated with alveolar inflammation, pulmonary infiltration
with monocytesmacrophages, progressive loss of alveolar structure and
pulmonary fibrosis [10]. The molecular mechanisms involved in the
pathogenesis of )IP have been elusive in spite of well-recognized
histologic and clinical manifestations.
[0006] There are two basic types of animals with genetically manipulated
genomes. A traditional transgenic mammal has a modified gene
introduced into its genome and the modified gene can be of exogenous
or endogenous origin. A "knockout" mammal is a special type of
transgenic mammal, characterized by suppression of the expression of
an endogenous gene through genetic manipulation. The disruption of
specific endogenous genes can be accomplished by deleting some
portion of the gene or replacing it with other sequences to generate a
null allele. Cross-breeding mammals having the null allele generates a
homozygous mammals lacking an active copy of the gene.
[0007] A number of such mammals have been developed, and are extremely
helpful in medical development. For example, U.S. Pat. No. 6,245,963,
details a knockout-transgenic mouse model of spinal muscular atrophy
and U.S. Pat. No. 6,414,219 details an osteopontin knockout mouse.
[0008] Transgenic animal models of SP-C mediated pulmonary diseases
would be very useful for identifying pharmaceutical agents that are
able to treat or prevent pulmonary diseases.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1. Progression of pulmonary histopathology in SP-C (-/-) mice.
Lungs were obtained from wild type littermates (A,C,E) or SP-C (-/-)
(B,D,F) mice. Lungs were inflation fixed at 20 cm H20 of pressure
and stained with hematoxylin-eosin. Airspace enlargement, variable
stromal thickening, and monocytic infiltration were noted at 2 months
(B), 6 months (D), and 12 months (F) of age. Perivascular and
peribrorichiolar mononuclear infiltrates and epithelial cell ~dysplasia in
conducting airways are shown in panel F. Micrographs 625x are
representative of at least 3-5 animals of similar ages.
[0010] Figure 2. Emphysema in SP-C (-/-) mice. Lung histology in control
(panel A) and SP-C (-/-) mice (panel B) demonstrate the marked
increase in alveolar size at one year of age. Macrophage infiltrates are
observed in the central and lower regions of panel B.
[0011] Figure 3. Remodeling and increased trichrome staining in lungs from
SP-C (-/-) mice. Mason trichrome (A,B), orcein (C,D), and a,-smooth
muscle actin immunostaining (E,F) are shown from lung tissue at 6
months of age in wild type (A,C,E) and SP-C (-/-) (B,D,F) littermates.
Airspace remodeling with monocytic infiltration and dense blue
staining was observed (arrows). Orcein staining demonstrated that
elastin fibers were absent in many of the remodeled airspaces (D). a-
Smooth muscle actin staining was observed in alveolar regions of the
lung parenchyma in SP-C (-/-) (arrows), but not wild type, mice.
[0012] Figure 4. Ultrastructural abnormalities in lungs of SP-C (-/-) mice.
Electronmicroscopy was performed on SP-C (-/-) mice at 9 months of
age. Marked abnormalities were observed in the alveolar walls from
the SP-C (-/-) mice (A). Type II cells were hyperplastic, containing
numerous lamellar body-like inclusions, collagen deposition was noted
within alveolar walls. Alveolar capillaries were surrounded by
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thickened subepithelial stroma. Conducting airways were lined by
dysplastic epithelial cells with atypical morphology (B). Numerous
cytopathic dense organelles, likely representing atypical mitochondria
were observed in nonciliated columnar epithelial cells. Alveolar
macrophages were hyperplastic, some containing dense crystals (top
cell, C). Others containing excessive amounts of surfactant lipids,
including lamellar bodies and tubular myelin figures, were observed.
Pulmonary vascular abnormalities were observed in small vessels in
SP-C (-/-) mice. Vessels were occluded or absent in many alveoli.
Abnormal membrane blebbing was recurrently observed along the
intima of the abnormal vessels (D).
[0013] Figure 5. Alveolar macrophage infiltrates in the SP-C (-/-) mice.
MAC-3 immunostaining was assessed in wild type (A) and SP-C (-/-)
(B) mice at 6 months of age. Extensive infiltration with MAC-3
staining cells was noted in association with severe emphysema (B).
Micrograph (625x) is representative of at least 5 SP-C (-/-) mice and
controls. Semi-thin sections of wild type (C) and SP-C (-/-) (D) mice
were stained with toluidine-blue, demonstrating alveolar and alveolar
macrophage abnormalities. Lipid inclusions were noted in hyperplastic
type II cells lining the alveoli and in the numerous alveolar
macrophages accumulating in the airspaces.
[0014] Figure 6. Epithelial cell dysplasia and MUCSA/C staining in
conducting airways of 2 month old SP-C (-l-) mice. Conducting
airways from wild type (A,C) or SP-C (-/-) (B,D) are observed after
H&E staining (A,B) ~or MUCSA/C immunohistochemistry (C,D).
Epithelial cell dysplasia was observed in large and small conducting '
airways of SP-C (-/-) mice. The abnormal epithelial cells were
hypertrophic with abnormal foci of pseudostratified epithelia. While
MUCSA/C staining cells were rarely seen in wild type mice (C),
extensive staining for MUCSA/C was observed throughout bronchi and
bronchioles (D), and was occasionally observed in the peripheral lung
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parenchyma in SP-C (-/-) mice (not shown). Panels A,B: 625x
magnification; Panels C,D: 1250x magnification.
[0015] Figure 7. Pressure-volume analysis demonstrates increased lung
volumes in SP-C (-/-) mice. Pressure-volume curves were performed
in tracheotomized wild type and SP-C (-/-) mice at 10-12 months of
age, n=5 per group. Significantly increased lung volumes at higher
pressure were observed in SP-C (-/-) mice, *p<0.01.
[0016] Figure 8. Phospholipid (SatPC) and surfactant proteins in SP-C (-/-)
mice. A: SatPC pool sizes were determined in wild type and SP-C (-/-
mice in BALF, lung tissue after BAL and the sum of BALF and tissue
fractions (total). SatPC were increased 60% in BALF and 2-fold in
tissue and total in SP-C (-/-) mice as compared to wild type mice at 15
months of age. B: Amounts of surfactant proteins in BALF were
estimated by Western blot relative to the amount of SatPC. Values for
wild type mice were normalized to a value of 1. SP-A and SP-D were
increased in SP-C (-/-) mice. C: Pool sizes/body weight for SP-A, SP-
B, and SP-D in BALF were normalized to a value of 1 for wild type
mice. While SP-B levels were unaltered, SP-A and SP-D were
increased in SP-C (-/-) mice. Mean ~ SE. *p<0.05.
[0017] Figure 9. Increased metalloproteinase activity produced by
macrophages from SP-C (-/-) mice. MMP activity was assessed by
zymography of conditioned media from alveolar macrophages from
SP-C (-/-), lane 1 and SP-C (+/+), lane 2. Protease activity 72 kd
(MMP-2) and 105 kd (MMP-9) were increased in media from SP-C (-/-
mice (arrows). A faint band at 55 kd, consistent with the size of
MMP-12, was also increased in conditional media from SP-C (-/-)
mice (arrowhead). Gels are consistent with observations from 4
separate experiments.
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STATEMENT OF THE INVENTION
[0018] The generation of SP-C (-/-) mice in a congenic 129JSV strain resulted
in the surprising finding that genetic ablation of SP-C caused a
progressive severe pulmonary fibrosis, expression of the mucin gene
MUCS in the conducting airways, epithelial cell dysplasia in
conducting airways, emphysema, alveolar vascular remodeling, and
right heart hypertrophy. Surprisingly, severe lung pathology developed
in the absence of associated abnormalities in surfactant concentrations,
and minimal alterations in surface properties of pulmonary surfactant
isolated from the lung of SP-C (-/-) mice were observed. These
findings demonstrate that a specific lack of SP-C/proSP-C per se,
causes severe lung disease.
[0019] In humans bearing dominantly inherited gene mutations in SP-C (that
causes the production of a misfolded proprotein, as well as disrupting
the expression of the normal protein), a deficiency of proSP-C or SP-C
per se also causes pulmonary disease. The pathology of the lung
disease includes idiopathic pulmonary fibrosis (IPF), desquamating
interstitial pneumonitis (DIP), usual interstitial pneumonitis (UIP),
non-specific interstitial pneumonitis (NSIP), and other forms of
interstitial lung disease.
[0020] The present invention provides for the use of a diagnostic screening
based on the absence of SP-C or proSP-C in tissues or lavage lung
material using immunohistochemistry, ELISA, Western blots, Mass
spectroscopy, and protein sequencing.
[0021] The present invention also provides for replacement of proSP-C or SP-
C, whether by gene transfer vectors to express the normal allele or
protein replacement with purified SP-C, proSP-C or recombinant SP-C
or recombinant proSP-C or SP-C or proSP-C analogues, are beneficial
for the treatment of these pulmonary disorders. SP-C may be
administered by aerosol or inhalation of a pharmaceutically useful
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preparation containing surfactant-like phospholipids, including
phosphatidylglycerol, phosphatidylcholine.
[0022] The present invention also provides for SP-C (-/-) mice providing a
model for testing therapies for interstitial lung disease, and for
determining molecular pathways, activated or suppressed, that
contribute to or cause the severe pulmonary disease seen in SP-C (-/-)
mice.
[0023] The present invention provides methods of producing a mouse with a
targeted disruption in a surfactant protein C (SP-C) gene. The present
invention also provides for a transgenic mouse produced by a targeted
disruption in a surfactmt protein C (SP-C) gene. The present invention
further provides for a cell or cell line from a transgenic mouse
produced by a targeted disruption in a surfactant protein C (SP-C)
gene. The present invention further provides for a surfactant protein C
knock-out construct, comprising a portion of an surfactant protein C
(SP-C) gene, wherein an internal portion of said SP-C gene is replaced
by a selectable marker and at least 50 consecutive nucleotides of SP-C
gene coding sequence have been deleted. Preferably, the SP-C deficient
(SP-C -/-) mice develop a severe progressive pulmonary disorder with
histologic features consistent with interstitial pneumonitis.
[0024] SP-C deficient mice developed severe, progressive pulmonary disease
associated with emphysema, diffuse alveolar fibrosis, monocytic
infiltrates, and epithelial cell dysplasia in conducting and peripheral
airways. Targeted deletion of proSP-C in mice causes a syndrome
similar to interstitial pneumonitis in humans.
[0025] In one aspect, the invention provides transgenic non-human organisms
and cell lines for use in the in vivo screening and evaluation of drugs or
other therapeutic regimens useful in the treatment of pulmonary
disorders. In one embodiment, the invention is a transgenic animal with
a targeted disruption in a pulmonary surfactant gene. In particular, the
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gene is the SP-C gene. The animal may be chimeric, heterozygotic or
homozygotic for the disrupted gene. Homozygotic knockout SP-C
mammals have a strong tendency towards developing a pulmonary
condition, such as emphysema, monocytic infiltrates, fibrosis,
epithelial cell dysplasia, and atypical accumulations of intracellular
lipids in type II epithelial cells and alveolar macrophages. The targeted
disruption may be anywhere in the gene, subject only to the
requirement that it inhibit production of functional SP-C protein.
[0026] The DNA sequence of the mouse surfactant protein C (SP-C) gene
(GenBank Acc. No. M38314) is shown in SEQ ID N0:1. The exonic
DNA sequence of the mouse SP-C gene (GenBank Acc. No. M38314)
is shown in SEQ m NO:2. The polypeptide sequence of the mouse
surfactant protein C (SP-C) (GenBanlc Acc. No. AAA40010) is shown
in SEQ m N0:3. The DNA sequence of the human surfactant protein
C (SP-C) gene (GenBanlc Acc. No. J03890) is shown in SEQ ID N0:4.
The polypeptide sequence of the human surfactant protein C (SP-C)
(GenBank Acc. No. AAC32022) is shown in SEQ m NO:S. The DNA
sequence of the human surfactant protein Cl (SP-C1) (GenBank Acc.
No. AAC32023) is shown in SEQ m N0:6.
[0027] In a preferred embodiment, the disruption occurs within exon 2 of the
wild type gene. In a more preferred embodiment, the disruption
includes at least a disruption of nucleotide 1667 at the ApaLl site in
exon 2 of the wild type gene. The transgenic animal may be of any
species (except human), but is preferably a mammal. In a preferred
embodiment, the non-human animal comprising a targeted disruption
in the surfactant protein C gene, wherein said targeted disruption
inhibits production of wild-type surfactant protein C so that the
phenotype of a non-human mammal homozygous for the targeted
disruption is characterized by a pulmonary disorder condition.
[0028] In another aspect, the invention features a cell or cell line, which
contains a targeted disruption in the surfactant protein C gene. In a
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preferred embodiment, the cell or cell line is am undifferentiated cell,
for example, a stem cell, embryonic stem cell, oocyte or embryonic
cell.
[0029] Yet in a further aspect, the invention features a method of producing a
non-human mammal with a targeted disruption in a surfactant protein
gene. For example, an SP-C knockout construct can be created with a
portion of the SP-C gene having an internal portion of said SP-C gene
replaced by a marker. The knockout construct can then be transfected
into a population of embryonic stem (ES) cells. Transfected cells can
then be selected as expressing the marker. The transfected ES cells can
then be introduced into an embryo of an ancestor of said mammal. The
embryo can be allowed to develop to term to produce a chimeric
mammal with the knockout construct in its germline. Breeding said
chimeric mammal will produce a heterozygous mammal with a
targeted disruption in the SP-C gene. Homozygotes can be generated
by crossing heterozygotes.
[0030] In another aspect, the invention features SP-C knockout constructs,
which can be used to generate the animals described above. In one
embodiment, the SP-C construct can comprise a portion of the
surfactant protein C (SP-C) gene, wherein an internal portion of the
gene is replaced by a selectable marker. Preferably, the marker is
neomycin resistance gene and the portion of the SP-C gene is at least
2.5 kb long or 7.0 or 9.5 kb long (including the replaced portion and
any SP-C flanking sequences). The internal portion preferably covers at
least a portion of an exon and most preferably it is at least nucleotide
1667 at the ApaLl site in exon 2 of the wild type gene.
[0031] In still another aspect, the invention features methods for testing
agents
for effectiveness in treating and/or preventing a pulmonary condition.
In one embodiment, the method can employ the transgenic animal or
cell lines, as described above. For example, a test agent can be
administered to the transgenic animal and the ability of the agent to
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ameliorate the pulmonary condition can be scored as having
effectiveness against said pulmonary condition. Any pulmonary
condition with a surfactant component can be tested using these
mammals, but in particular, conditions characterized by a lack of SP-C
protein are studied. The method may also be used to test agents that are
effective in replacing the SP-C pulmonary proteins and their
downstream components.
[0032] Unless otherwise defined, all technical and scientific terms used
herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. All publications, patent applications,
patents, and other references mentioned herein are specifically
incorporated by reference in their entirety. In the case of conflict, the
present specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and not
intended to be limiting.
[0033] Other features and advantages of the invention will be apparent from
the following detailed description and claims.
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DETAILED DESCRIPTION OF THE INVENTION
[0034] In the description that follows, a number of terms used in recombinant
DNA technology are extensively utilized. In order to provide a clear
and consistent understanding of the specification and claims, including
the scope to be given such terms, the following definitions are
provided.
[0035] The term "agonist", as used herein, is meant to refer to an agent that
mimics or upregulates (e.g. potentiates or supplements) SP-C
bioactivity. An SP-C agonist can be a wild-type SP-C protein or
derivative thereof having at least one bioactivity of the wild-type SP-C.
An SP-C therapeutic can also be a compound that upregulates
expression of an SP-C gene or which increases at least one bioactivity
of the SP-C protein. Agonists can be any class of molecule, preferably
a small molecule, including a nucleic acid, protein, carbohydrate, lipid
or combination thereof.
[0036] "Antagonist" as used herein is meant to refer to an agent that down-
regulates (e.g. suppresses or inhibits) at least one SP-C bioactivity. An
antagonist can be a compound that down-regulates expression of an
SP-C locus gene or that reduces the amount of an SP-C protein present.
The SP-C antagonist can also be an SP-C antisense nucleic acid or a
ribozyme capable of interacting specifically with SP-C RNA. Yet other
SP-C antagonists are molecules that bind to SP-C polypeptide and
inhibit its action. Such molecules include peptides. Yet other SP-C
antagonists include antibodies interacting specifically with an epitope
of an SP-C molecule, such that binding interferes with the biological
function of the SP-C locus polypeptide.
[0037] The term "allele", which is used interchangeably herein with "allelic
variant" refers to alternative forms of a gene or portions thereof.
Alleles occupy the same locus or position on homologous
chromosomes. When a subject has two identical alleles of a gene, the
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subject is said to be homozygous for the gene or allele. When a subject
has two different alleles of a gene, the subject is said to be
heterozygous for the gene or allele. Alleles of a specific gene can differ
from each other in a single nucleotide, or several nucleotides, and can
include substitutions, deletions, and insertions of nucleotides.
Frequently occurring sequence variations include transition mutations
(i.e. purine to purine substitutions and pyrimidine to pyrimidine
substitutions, e.g. A to G or C to T), transversion mutations (i.e. purine
to pyrimidine and pyrimidine to purine substitutions, e.g. A to T or C
to G), and alteration in repetitive DNA sequences (e.g. expansions and
contractions of trinucleotide repeat and other tandem repeat
sequences). An allele of a gene can also be a form of a gene containing
a mutation. The term "allelic variant of a polymorphic region of an SP-
C gene" refers to a region of an SP-C locus gene having one or several
nucleotide sequence differences found in that region of the gene in
other individuals.
[0038] As used herein, "pulmonary disease" refers to disorders and conditions
generally recognized by those skilled in the art as related to the
constellation of pulmonary diseases characterized by emphysema,
monocytic infiltrates, fibrosis, epithelial cell dysplasia, and atypical
accumulations of intracellular lipids in type II epithelial cells and
alveolar macrophages, regardless of the cause or etiology. These
include, but are not limited to, emphysema and interstitial pneumonitis.
[0039] "Biological activity" or "bioactivity" or "activity" or "biological
function", which are used interchangeably, for the purposes herein
means a function that is directly or indirectly performed by an SP-C
polypeptide (whether in its native or denatured conformation), or by
any subsequence thereof. SP-C bioactivity can be modulated by
directly affecting an SP-C polypeptide. Alternatively, an SP-C
bioactivity can be modulated by modulating the level of an SP-C
polypeptide, such as by modulating expression of an SP-C gene.
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[0040] As used herein the term "bioactive fragment of an SP-C polypeptide"
refers to a fragment of a full-length SP-C polypeptide, wherein the
fragment specifically mimics or antagonizes the activity of a wild-type
SP-C polypeptide.
[0041] The term "aberrant activity", as applied to an activity of a
polypeptide
such as SP-C, refers to an activity which differs from the activity of the
wild-type or native polypeptide or which differs from the activity of the
polypeptide in a healthy subject. An activity of a polypeptide can be
aberrant because it is stronger than the activity of its native counterpart.
Alternatively, an activity can be aberrant because it is weaker or absent
relative to the activity of its native counterpart. An aberrant activity can
also be a change in an activity. A cell can have an aberrant SP-C
activity due to overexpression or underexpression of an SP-C locus
gene encoding an SP-C locus polypeptide.
[0042] "Cells", "host cells" or "recombinant host cells" are terms used
interchangeably herein. It is understood that such terms refer not only
to the particular subject cell but to the progeny or potential progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[0043] A "chimeric polypeptide" or "fusion polypeptide" is a fusion of a first
amino acid sequence encoding one of the subj ect SP-C locus
polypeptides with a second amino acid sequence defining a domain
(e.g. polypeptide portion) foreign to and not substantially homologous
with any domain of an SP-C polypeptide. A chimeric polypeptide may
present a foreign domain that is found (albeit in a different
polypeptide) in an organism that also expresses the first polypeptide, or
it may be an "interspecies", "intergenic", etc. fusion of polypeptide
structures expressed by different kinds of organisms. In general, a
fusion polypeptide can be represented by the general formula X-SP-C-
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Y, wherein SP-C represents a portion of the polypeptide that is derived
from an SP-C polypeptide, and X and Y are independently absent or
represent amino acid sequences that are not related to an SP-C
sequence in an organism, including naturally occurring mutants.
[0044] The phrase "nucleotide sequence complementary to the nucleotide
sequence set forth in SEQ ID NO. x" refers to the nucleotide sequence
of the complementary strand of a nucleic acid strand having SEQ m
NO. x. The term "complementary strand" is used herein
interchangeably with the term "complement". The complement of a
nucleic acid strand can be the complement of a coding strand or the
complement of a non-coding strand. When referring to double stranded
nucleic acids, the complement of a nucleic acid having SEQ m NO. x
refers to the complementary strand of the strand having SEQ ID NO. x
or to any nucleic acid having the nucleotide sequence of the
complementary strand of SEQ ID NO. x. When referring to a single
stranded nucleic acid having the nucleotide sequence SEQ m NO. x,
the complement of this nucleic acid is a nucleic acid having a
nucleotide sequence which is complementary to that of SEQ ID NO. x.
The nucleotide sequences and complementary sequences thereof are
always given in the 5' to 3' direction.
[0045] As is well known, genes may exist in single or multiple copies within
the genome of an individual. Such duplicate genes may be identical or
may have certain modifications, including nucleotide substitutions,
additions or deletions, which all still code for polypeptides having
substantially the same activity. The term "DNA sequence encoding an
SP-C polypeptide" may thus refer to one or more genes within a
particular individual. Moreover, certain differences in nucleotide
sequences may exist between individual organisms, which are called
alleles. Such allelic differences may or may not result in differences in
amino acid sequence of the encoded polypeptide yet still encode a
polypeptide with the same biological activity.
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[0046] The phrases "disruption of the gene" and "targeted disruption" or any
similar phrase refers to the site specific interruption of a native DNA
sequence so as to prevent expression of that gene in the cell as
compared to the wild-type copy of the gene. The interruption may be
caused by deletions, insertions or modifications to the gene, or any
combination thereof.
[0047] The term "haplotype" refers to a set of alleles that are inherited
together as a group (are in linkage disequilibrium). As used herein,
haplotype is defined to include those haplotypes that occur at
statistically significant levels (p<0.05). As used herein, the phrase "an
SP-C haplotype" refers to a haplotype in the SP-C locus.
[0048] "Homology" or "identity" or "similarity" refers to sequence similarity
between two peptides or between two nucleic acid molecules.
Homology can be determined by comparing a position in each
sequence that may be aligned for purposes of comparison. When a
position in the compared sequence is occupied by the same base or
amino acid, then the molecules are identical at that position. A degree
of homology or similarity or identity between nucleic acid sequences is
a function of the number of identical or matching nucleotides at
positions shared by the nucleic acid sequences. A degree of identity of
amino acid sequences is a function of the number of identical amino
acids at positions shared by the amino acid sequences. A degree of
homology or similarity of amino acid sequences is a function of the
number of amino acids, i.e. structurally related, at positions shared by
the amino acid sequences. An "unrelated" or "non-homologous"
sequence shares less than 40% identity, though preferably less than
25% identity, with one of the SP-C locus sequences of the present
invention.
[0049] The term "interact" as used herein is meant to include detectable
relationships or association (e.g. biochemical interactions) between
molecules, such as interaction between protein-protein, protein-nucleic
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acid, nucleic acid-nucleic acid, and protein-small molecule or nucleic
acid-small molecule in nature.
[0050] The term "SP-C related" as used herein is meant to include all mouse
and human genes related to the human SP-C locus genes on human
chromosome 8.
[0051] Where the term "SP-C" is used in reference to a gene product or
polypeptide, it is meant to refer to all gene products encoded by the
surfactant protein C locus on human chromosome 8 and their
corresponding mouse homologs.
[0052] The term "SP-C therapeutic" refers to various forms of SP-C
polypeptides, as well as peptidomimetics, nucleic acids, or small
molecules, which can modulate at least one activity of an SP-C
polypeptide by mimicking or potentiating (agonizing) or inhibiting
(antagonizing) the effects of a naturally-occurring SP-C polypeptide.
An SP-C therapeutic that mimics or potentiates the activity of a wild-
type SP-C polypeptide is a "SP-C agonist". Conversely, an SP-C
therapeutic that inhibits the activity of a wild-type SP-C polypeptide is
an "SP-C antagonist".
[0053] The term "isolated" as used herein with respect to nucleic acids, such
as DNA or RNA, refers to molecules separated from other DNAs, or
RNAs, respectively, that are present in the natural source of the
macromolecule. For example, an isolated nucleic acid encoding one of
the subject SP-C polypeptides preferably includes no more than 5
kilobases (kb) of nucleic acid sequence which naturally immediately
flanks the SP-C gene in genomic DNA, more preferably no more than
kb of such naturally occurring flanking sequences, and most
preferably less than 5 kb of such naturally occurring flanking sequence.
The term isolated as used herein also refers to a nucleic acid or peptide
that is substantially free of cellular material, viral material, or culture
medium when produced by recombinant DNA techniques, or chemical
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precursors or other chemicals when chemically synthesized. Moreover,
an "isolated nucleic acid" is meant to include nucleic acid fragments
that are not naturally occurnng as fragments and would not be found in
the natural state. The term "isolated" is also used herein to refer to
polypeptides that are isolated from other cellular proteins and is meant
to encompass both purified and recombinant polypeptides.
[0054] The term "knockout " refers to partial or complete suppression of the
expression of an endogenous gene. This is generally accomplished by
deleting a portion of the gene o~ by replacing a portion with a second
sequence, but may also be caused by other modifications to the gene
such as the introduction of stop codons, the mutation of critical amino
acids, the removal of an intron junction, etc.
[0055] The term "knockout construct" refers to a nucleic acid sequence that
can be used to decrease or suppress expression of a protein encoded by
endogenous DNA sequences in a cell. In a simple example, the
knockout construct is comprised of a gene, such as the SP-C gene, with
a deletion in a critical portion of the gene so that active protein cannot
be expressed therefrom. Alternatively, a number of termination codons
can be added to the native gene to cause early termination of the
protein or an intron junction can be inactivated. In a typical knockout
construct, some portion of the gene is replaced with a selectable marker
(such as the neo gene) so that the gene can be represented as follows:
SP-C 5'/neo/SP-C 3', where SP-CS' and SP-C 3', refer to genomic or
cDNA sequences which are, respectively, upstream and downstream
relative to a portion of the SP-C gene and where neo refers to a
neomycin resistance gene. In another knockout construct, a second
selectable marker is added in a flanking position so that the gene can be
represented as: SP-C/neo/SP-C/TK, where TK is a thymidine kinase
gene-which can be added to either the SP-CS' or the SP-C3' sequence
of the preceding construct and which further can be selected against
(i.e. is a negative selectable marker) in appropriate media. This two-
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marker construct allows the selection of homologous recombination
events, which removes the flanking TK marker, from non-homologous
recombination events that typically retain the TK sequences. The gene
deletion and/or replacement can be from the exons, introns, especially
intron junctions, and/or the regulatory regions such as promoters.
[0056] The term "knockout mammal" and the like, refers to a transgenic
mammal wherein a given gene has been suppressed by recombination
with a knockout construct. It is to be emphasized that the term is
intended to include all progeny generations. Thus, the founder animal
and all F1, F2, F3, and so on, progeny thereof are included.
[0057] The term "chimera," "mosaic," "chimeric mammal" and the like, refers
to a transgenic mammal with a knockout construct in some of its
genome-containing cells.
[0058] The term "heterozygote" "heterozygotic mammal" and the like, refers
to a transgenic mammal with a knockout construct on one of a
chromosome pair in all of its genome-containing cells.
[0059] The term "homozygote" "homozygotic mammal" and the like, refers to
a transgenic mammal with a knockout construct on both members of a
chromosome pair in all of its genome-containing cells.
[0060] "Linkage disequilibrium" refers to co-inheritance of two alleles at
frequencies greater than would be expected from the separate
frequencies of occurrence of each allele in a given control population.
The expected frequency of occurrence of two alleles that are inherited
independently is the frequency of the first allele multiplied by the
frequency of the second allele. Alleles that co-occur at expected
frequencies are said to be in "linkage equilibrium".
[0061] The term "marker" or "marker sequence" or similar phrase means any
gene that produces a selectable genotype or preferably a selectable
phenotype. It includes such examples as the neo gene, green
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fluorescent protein (GFP) gene, TK gene, ,~-galactosidase gene, etc.
The marker sequence may be any sequence known to those skilled in
the art that serves these purposes, although typically the marker
sequence will be a sequence encoding a protein that confers a
selectable trait, such as an antibiotic resistance gene, or an enzyme that
can be detected and that is not typically found in the cell. The marker
sequence may also include regulatory regions such as a promoter or
enhancer that regulates the expression of that protein. However, it is
also possible to transcribe the marker using endogenous regulatory
sequences. In one embodiment of the present invention, the marker
facilitates separation of transfected from untransfected cells by
fluorescence activated cell sorting, for example by the use of a
fluorescently labeled antibody or the expression of a fluorescent
protein such as GFP. Other DNA sequences that facilitate expression
of marker genes may also be incorporated into the DNA constructs of
the present invention. These sequences include, but are not limited to
transcription initiation and termination signals, translation signals,
post-translational modification signals, intron splicing junctions,
ribosome binding sites, and polyadenylation signals, to name a few.
The marker sequence may also be used to append sequence to the
target gene. For example, it may be used to add a stop codon to
truncate SP-C translation.
[0062] The use of selectable markers is well known in the art and need not be
detailed herein. The term "modulation" as used herein refers to both
upregulation (i.e., activation or stimulation (e.g., by agonizing or
potentiating)) and downregulation (i.e. inhibition or suppression (e.g.,
by antagonizing, decreasing or inhibiting)).
[0063] The term "mutated gene" refers to an allelic form of a gene, which is
capable of altering the phenotype of a subject having the mutated gene
relative to a subject that does not have the mutated gene. If a subject
must be homozygous for this mutation to have an altered phenotype,
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the mutation is said to be recessive. If one copy of the mutated gene is
sufficient to alter the genotype of the subject, the mutation is said to be
dominant. If a subject has one copy of the mutated gene and has a
phenotype that is intermediate between that of a homozygous and that
of a heterozygous subject (for that gene), the mutation is said to be co-
dominant.
[0064] The "non-human animals" of the invention include mammalians such
as rodents, non-human primates, sheep, dog, cow, chickens,
amphibians, reptiles, etc. Preferred non-human animals are selected
from the rodent family including rat and mouse, most preferably
mouse, though transgenic amphibians, such as members of the
Xenopus genus, and transgenic chickens can also provide important
tools for understanding and identifying agents which can affect, for
example, embryogenesis and tissue formation. The term "chimeric
animal" is used herein to refer to animals in which the recombinant
gene is found, or in which the recombinant gene is expressed in some
but not all cells of the animal. The term "tissue-specific chimeric
animal" indicates that one of the recombinant SP-C genes is present
and/or expressed or disrupted in some tissues but not others. The teen
"non-human mammal" refers to any members of the class Mammalia,
except for humans.
[0065] As used herein, the term "nucleic acid" refers to polynucleotides or
oligonucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as equivalents, analogs of either RNA or DNA
made from nucleotide analogs and as applicable to the embodiment
being described, single (sense or antisense) and double-stranded
polynucleotides.
[0066] The teen "polymorphism" refers to the coexistence of more than one
form of a gene or portion (e.g., allelic variant) thereof A portion of a
gene of which there are at least two different forms, i.e., two different
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nucleotide sequences, is referred to as a "polymorphic region of a
gene". A polymorphic region can be a single nucleotide, the identity of
which differs in different alleles. A polymorphic region can also be
several nucleotides long.
[0067] A "polymorphic gene" refers to a gene having at least one polymorphic
region.
[006] As used herein, the term "promoter" means a DNA sequence that
regulates expression of a selected DNA sequence operably linked to the
promoter, and which effects expression of the selected DNA sequence
in cells. The term encompasses "tissue specific" promoters, i:e.
prom~ters, which effect expression of the selected DNA sequence only
in specific cells (e.g. cells of a specific tissue). The term also covers so-
called "leaky" promoters, which regulate expression of a selected DNA
primarily in one tissue, but cause expression in other tissues as well.
The teen also encompasses non-tissue specific promoters and
promoters that constitutively express or that are inducible (i. e.
expression levels can be controlled).
[0069] The terms "protein", "polypeptide"'and "peptide" are used
interchangeably herein when referring to a gene product.
[0070] The term "recombinant protein" refers to a polypeptide of the present
invention which is produced by recombinant DNA techniques, wherein
generally, DNA encoding an SP-C polypeptide is inserted into a
suitable expression vector which is in turn used to transform a host cell
to produce the heterologous protein. Moreover, the phrase "derived
from", with respect to a recombinant SP-C gene, is meant to include
within the meaning of "recombinant protein" those proteins having~an
amino acid sequence of a native SP-C polypeptide, or an amino acid
sequence similar thereto which is generated by mutations including
substitutions and deletions (including truncation) of a naturally
occurring form of the polypeptide.
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[0071] "Small molecule" as used herein, is meant to refer to a composition,
which has a molecular weight of less than about 5 kD and most
preferably less than about 4 kD. Small molecules can be nucleic acids,
peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other
organic (carbon containing) or inorganic molecules. Many
pharmaceutical companies have extensive libraries of chemical and/or
biological mixtures, often fungal, bacterial, or algal extracts, which can
be screened with any of the assays of the invention to identify
compounds that modulate an SP-C bioactivity.
[0072] As used herein, the term "specifically hybridizes" or "specifically
detects" refers to the ability of a nucleic acid molecule of the invention
to hybridize to at least approximately 6, 12, 20, 30, 50, 100, 150, 200,
300, 350, 400 or 425 consecutive nucleotides of a vertebrate,
preferably an SP-C gene.
[0073] "Transcriptional regulatory sequence" is a generic term used
throughout the specification to refer to DNA sequences, such as
initiation signals, enhancers, and promoters, which induce or control
transcription of protein coding sequences with which they are operably-
linked. In preferred embodiments, transcription of one of the SP-C
genes is under the control of a promoter sequence (or other
transcriptional regulatory sequence) that controls the expression of the
recombinant gene in a cell-type in which expression is intended. It will
also be understood that the recombinant gene can be under the control
of transcriptional regulatory sequences which are the same or which are
different from those sequences which control transcription of the
naturally-occurring forms of SP-C polypeptide.
[0074] As used herein, the term "transfection" means the introduction of a
nucleic acid, e.g., via an expression vector, into a recipient cell by
nucleic acid-mediated gene transfer. Methods for transformation that
are known in the art include any electrical, magnetic, physical,
biological or chemical means. As used herein, "transfection" includes
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such specific techniques as electroporation, magnetoporation, Ca++
treatment, injection, bombardment, retroviral infection and lipofection,
among others. "Transformation", as used herein, refers to a process in
which a cell's genotype is changed as a result of the cellular uptake of
exogenous DNA or RNA, and, for example, the transformed cell
expresses a recombinant form of an SP-C polypeptide or, in the case of
anti-sense expression from the transferred gene, the expression of a
naturally-occurring form of the SP-C polypeptide is disrupted.
[0075] As used herein, the term "transgene" means a nucleic acid sequence
(encoding, e.g., one of the SP-C polypeptides, or an antisense transcript
thereto) that has been introduced into a cell. A transgene could be
partly or entirely heterologous, i. e., foreign, to the transgenic animal or
cell into which it is introduced, or, is homologous to an endogenous
gene of the transgenic animal or cell into which it is introduced, but
which is designed to be inserted, or is inserted, into the animal's
genome in such a way as to alter the genome of the cell into which it is
inserted (e.g., it is inserted at a location which differs from that of the
natural gene or its insertion results in a knockout). A transgene can also
be present in a cell in the form of an episome. A transgene can include
one or more transcriptional regulatory sequences and any other nucleic
acid, such as introns, that may be necessary for optimal expression of a
selected nucleic acid.
[0076] A "transgenic animal" refers to any animal, preferably a non-human
mammal, bird or an amphibian, in which one or more,of the cells of the
a~.umal contain heterologous nucleic acid introduced by way of human
intervention, such as by transgenic techniques well known in the art.
The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell, by way of deliberate genetic
manipulation, such as by microinjection or by infection with a
recombinant virus. The term genetic manipulation does not include
classical cross-breeding, or in vitro fertilization, but rather is directed
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to the introduction of a recombinant DNA molecule. This molecule
may be integrated within a chromosome, or it may be
extrachromosomally replicating DNA. In the typical transgenic animals
described herein, the transgene causes cells to express a recombinant
form of one of the SP-C polypeptides, e.g. either agonistic or
antagonistic forms. However, transgenic animals in which the
recombinant SP-C gene is silent are also contemplated, as for example,
the FLP or CRE recombinase dependent constructs described below.
Moreover, "transgenic animal" also includes those recombinant
animals in which gene disruption of one or more SP-C genes is caused
by human intervention, including both recombination and antisense
techniques.
[0077] The term "treating" as used herein is intended to encompass curing as
well as ameliorating at least one symptom of the condition or disease.
[0078] The term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type
of preferred vector is an episome, i.e., a nucleic acid capable of extra-
chromosomal replication. Preferred vectors are those capable of
autonomous replication and/or expression of nucleic acids to which
they are linked. Vectors capable of directing the expression of genes to
which they are operatively linked are referred to herein as "expression
vectors". In general, expression vectors of utility iri recombinant DNA
techniques are often in the form of "plasmids" which refer generally to
circular double stranded DNA loops which, in their vector form are not
bound to the chromosome. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most commonly
used form of vector. However, the invention is intended to include
such other forms of expression vectors which serve equivalent
functions and which become known in the art subsequently hereto.
[0079] The term "wild-type allele" refers to an allele of a gene which, when
present in two copies in a subject results in a wild-type phenotype.
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There can be several different wild-type alleles of a specific gene, since
certain nucleotide changes in a gene may not affect the phenotype of a
subj ect having two copies of the gene with the nucleotide changes.
[0080] The present invention provides for a method of treating pulmonary
disease in a subj ect comprising the administration to a subj ect in need
of such treatment a therapeutically effective amount of a formulation
comprising a SP-C therapeutic. Preferably, the SP-C therapeutic is an
agent selected from the group consisting of an isolated SP-C protein,
an isolated nucleic acid molecule encoding a SP-C protein, a SP-C
receptor-specific antibody that stimulates the activity of the receptor, or
pharmaceutically acceptable composition thereof.
[0081] The present invention provides for the use of a SP-C therapeutic agent
wherein the agent is a SP-C receptor-specific antibody that stimulates
the activity of the receptor or wherein the agent is an isolated SP-C
protein or proSP-C protein.
[0082] In one embodiment, the SP-C therapeutic agent is an isolated nucleic
acid molecule encoding a SP-C protein or proSP-C protein, wherein
the nucleic acid molecule is operatively linked to a transcription
control sequence. Preferably, the nucleic acid molecule is expressed in
the subj ect's airway cells. More preferably, the nucleic acid encodes a
SP-C polypeptide, fragment, homolog or variant with substantial
homology, supplying SP-C function.
[0083] In one embodiment, the nucleic acid molecule becomes integrated to
the chromosomal DNA making up the genome of the subject's airway
cells. In another embodiment, the nucleic acid molecule is expressed
by the subject's airway cells from an extrachromosomal location.
Generally, the nucleic acid molecule comprises at least 50 nucleotides.
Preferably, the nucleic acid molecule comprises at least 200
nucleotides. The airway cells are generally smooth muscle and
epithelial cells.
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[0084] In one embodiment, the isolated nucleic acid molecule is administered
to the mammal complexed with a liposome delivery vehicle.
Alternatively, the isolated nucleic acid molecule is administered to the
mammal in a viral vector delivery vehicle. Preferably, the viral vector
delivery vehicle is from adenovirus.
[0085] In one embodiment, the isolated nucleic acid molecule, when
administered to the lungs of the mammal, is expressed in cells of the
mammal. Preferably, the disease is a chronic obstructive pulmonary
disease of the airways associated with eosinophilic inflammation.
[0086] In another embodiment, the disease is selected from the group
consisting of airway obstruction, allergies, asthma, acute inflammatory
lung disease, chronic inflammatory lung disease, chronic obstructive
pulmonary dysplasia, emphysema, pulmonary emphysema, chronic
obstructive emphysema, adult respiratory distress syndrome,
bronchitis, chronic bronchitis, chronic asthmatic bronchitis, chronic
obstructive bronchitis, and interstitial lung diseases.
[0087] Preferably, the SP-C therapeutic agent decreases lung inflammation in
the mammal. The SP-C therapeutic agent is administered in an amount
between about 0.1 micrograms/kilogram and about 100
milligram/kilogram body weight of a mammal. Preferably in an amount
between about 0.1 micrograms/kilogram and about 10
milligram/kilogram body weight of a mammal. In one embodiment, the
SP-C therapeutic agent is administered in a pharmaceutically
acceptable excipient.
[0088] The SP-C therapeutic agent may be administered by at least one route
selected from the group consisting of nasal and inhaled routes.
[0089] In another embodiment, the pulmonary disease is selected from the
group consisting of asthma, allergic bronchopulmonary aspergillosis,
hypersensitivity pneumonia, eosinphilic pneumonia, allergic bronchitis
bronchiectasis, hypersensitivity pneumotitis, occupational asthma,
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reactive airway disease syndrome, hypereosinophilic syndrome,
rhinitis, sinusitis, and parasitic lung disease.
[0090] The present invention also provides for a method for prescribing
treatment for airway hyperresponsiveness and/or airflow limitation
associated with a respiratory disease involving an inflammatory
response in a mammal, comprising: a. administering to the lungs of a
mammal a SP-C therapeutic agent selected from the group consisting
of: a SP-C receptor-specific antibody that stimulates the activity of the
receptor an isolated SP-C protein or proSP-C protein; and an isolated
nucleic acid molecule encoding a SP-C protein or proSP-C protein,
wherein the nucleic acid molecule is operatively linked to a
transcription control sequence; b. measuring a change in lung function
in response to a provoking agent in the mammal to determine if the SP-
C therapeutic agent modulates airway hypenesponsiveness; and c.
prescribing a pharmacological therapy comprising administration of
SP-C therapeutic agent to the mammal effective to reduce
inflammation based upon the changes in lung function.
[0091] The present invention also provides for a formulation for protecting a
mammal from airway hyperresponsiveness, airflow limitation and/or
airway fibrosis associated with a respiratory disease involving
inflammation, comprising an anti-inflammatory agent effective for
reducing eosinophilic inflammation and a SP-C therapeutic agent
selected from the group consisting of: a SP-C receptor-specific
antibody that stimulates the activity of the receptor; an isolated SP-C
protein or proSP-C protein; and an isolated nucleic acid molecule
encoding a SP-C protein or proSP-C protein, wherein the nucleic acid
molecule is operatively linked to a transcription control sequence.
Generally, the formulation comprises a pharmaceutically acceptable
excipient.
[0092] Preferably, the formulation comprises a controlled release vehicle
selected from the group consisting of biocompatible polymers, other
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polymeric matrices, capsules, microcapsules, microparticles, bolus
preparations, osmotic pumps, diffusion devices; liposomes,
lipospheres, viral vectors and transdermal delivery systems.
[0093] In one embodiment, the SP-C therapeutic agent is an isolated SP-C
protein or proSP-C protein. In another, the SP-C therapeutic agent is an
isolated nucleic acid molecule encoding a SP-C protein or proSP-C
protein, wherein the nucleic acid molecule is operatively linked to a
transcription control sequence. In one embodiment, the isolated nucleic
acid molecule is complexed with a liposome delivery vehicle. In
another embodiment, the isolated nucleic acid molecule is provided in
a viral vector delivery vehicle. Preferably, the viral vector delivery
vehicle is from adenovirus.
[0094] In another embodiment, the SP-C therapeutic agent is a SP-C receptor-
specific antibody that stimulates the activity of the receptor. In another
embodiment, the SP-C therapeutic agent is selected from the group
consisting of: an isolated SP-C protein or proSP-C protein and an
isolated nucleic acid molecule encoding a SP-C protein or proSP-C
protein, wherein the nucleic acid molecule is operatively linked to a
transcription control sequence.
[0095] In another embodiment, the formulation may contain an anti-
inflammatory agent selected from the group consisting of anti-IgE,
immunomodulating drugs, leukotriene synthesis inhibitors, leukotriene
receptor antagonists, glucocorticosteroids, steroid chemical derivatives,
anti-cyclooxygenase agents, beta-adrenergic agonists, methylxanthines,
cromones, anti-CD4 reagents, anti-IL-5 reagents, surfactants, cytoxin,
and heparin. Preferably, anti-inflammatory agent is selected from the
group consisting of leukotriene synthesis inhibitors, leukotriene
receptor antagonists, glucocorticosteroids, beta-adrenergic agonists,
methylxanthines, and cromones.
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[0096] In general, the invention provides transgenic animals in which one or
more SP-C related genes have been modified by transgenic cloning
procedures. These SP-C transgenic animals are useful as animal
models for various diseases that involve SP-C mediated pulmonary
processes. Although most of the above described pulmonary diseases
and conditions appear to have a complex and multifactorial etiology,
they all appear to ultimately involve SP-C mediated pulmonary
processes. The present invention also provides reagents and methods
for the discovery of pharmaceutical compounds that are able to
interfere with these SP-C mediated pulmonary processes and thereby
block the progression of these otherwise disparate diseases.
[0097] In a preferred embodiment, the invention features a transgenic
"knockout" mouse line in which the mouse SP-C (surfactant protein C)
gene carried on mouse chromosome 14 at position 8p,8 is disrupted or
deleted so as to decrease or eliminate expression of the SP-C gene.
[0098] The SP-C knockout mouse line features an enhancement of SP-C-
mediated pulmonary disorder processes due to loss of endogenous SP-
C protein molecules. In a further embodiment, the invention provides a
"double" knockout mouse line featuring decreased expression of both
the SP-C gene and at least one gene of SP-A, SP-B, or SP-D.
[0099] The transgenic "knockout" mouse line is useful to generate both
heterozygous and homozygous SP-C gene knockout mice which can be
used to study SP-C mediated pulmonary diseases and conditions. For
example, loss of the surfactant protein C protein leads to increased SP-
C mediated pulmonary disorders, and this contributes to the etiology of
a number of diseases and conditions including: emphysema, monocytic
infiltrates, fibrosis, epithelial cell dysplasia, and atypical accumulations
of intracellular lipids in type II epithelial cells and alveolar
macrophages.
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[00100] Both chronic and acute forms of such pulmonary diseases and
conditions can be reproduced in appropriate SP-C knockout animals or
animal lines. For example, animals heterozygous for the SP-C
knockout construct have diminished capacity to produce the surfactant
protein C and therefore show a corresponding accentuation of SP-C
mediated pulmonary processes. The heterozygous mouse lines may
therefore reproduce the circumstances of chronic pulmonary diseases
and conditions. Furthermore, these heterozygous animals or cell lines
are well suited to finding therapeutic agents which act to accentuate the
expression or activity of the diminished pool of endogenous surfactant
protein C. Such receptor antagonist "agonists" may, for example,
increase expression of the remaining copy of the SP-C gene. In
contrast, homozygous SP-C "knockout" animals and lines have no
ability to produce the SP-C gene product and hence show a
correspondingly large enhancement of SP-C mediated processes. The
homozygous animals and cell lines may therefore reproduce the
aberrant pulmonary functions that occur in acute pulmonary diseases.
Furthermore, these homozygous animals and lines are especially well
suited to fording therapeutic agents that function, for example, as
molecular mimics of the surfactant protein C by, for example.
[00101] The invention further provides various nucleic acid constructs useful
for creating SP-C "knockout" and SP-C "knock-in" transgenic mouse
cell lines and transgenic mice.
[00102] For example, an SP-C disrupting construct can be engineered so as to
incorporate a reporter or marker gene (such as beta-galactosidase or
green fluorescent protein) into a chromosomal copy of the gene,
thereby rendering the resulting chimeric reporter gene dependent upon
the endogenous SP-C gene promoter for its expression. Transgenic cell
lines and animals incorporating such "knock-in" constructs are
particularly well suited to the screening of compounds for their ability
to suppress SP-C dependent pulmonary processes by increasing the
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transcription of the surfactant protein C gene. In another example, a
heterologous regulatable promoter can be "knocked-in" to the SP-C
gene locus so that surfactant protein C expression is now controlled by
the regulatable promoter. The regulatable promoter can be an inducible
promoter, a repressible promoter or a developmentally regulated
promoter. The choice of promoters in this instance can be tailored to
the specific study at hand. For example, a repressible promoter system
facilitates the production of mouse lines in which the SP-C gene is
expressed until some point in time after normal growth and
development. The function of the SP-C gene can then be abruptly
halted by administration of an appropriate ligand (such as tetracycline)
which results in the transcriptional shut-down of the SP-C gene. This
inducible surfactant protein C deficiency thereby triggers SP-C
mediated pulmonary conditions in an otherwise normally developing
animal. Pharmaceutical screens can thus be devised for compounds
capable of blocking an surfactant protein C deficiency-induced
pulmonary response.
[00103] The SP-C transgenic animals and cell lines of the present invention
may thus be used for the development of pharmaceutical agents which
are useful for treating or preventing such SP-C mediated diseases and
conditions.
[00104] The gene to be knocked out may be any gene involved in the SP-C
pathway, provided that at least some sequence or mapping information
on the DNA to be disrupted is available to use in the preparation of
both the knockout construct and the screening probes. In a preferred
embodiment of the invention, the mouse SP-C gene on chromosome 14
at position 8p,8 is targeted for disruption. The genomic DNA sequence
of the marine SP-C gene is shown in SEQ )D NO:1. These target gene
constructs include SP-C target gene knockout and knock-in constructs
which are specifically adapted to each of the various embodiments of
the invention.
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[00105] Important aspects of the present invention concern the disruption of
genes, that express one or more SP-C polypeptides, generally having
the sequences of mouse (GenBanlc accession number M38314; SEQ ID
NO:1) or human (GenBank accession number J03~90; SEQ ID N0:4)
SP-C genes, or functional equivalents thereof. "Genes" refers to a
DNA segment including any of the SP-C gene coding sequences and,
in certain aspects, regulatory sequences, isolated substantially away
from other naturally occurring genes or protein encoding sequences. In
this respect, the term "gene" is used for simplicity to refer to a
functional protein, polypeptide or peptide encoding unit. As will be
understood by those in the art, this functional term includes both
genomic sequences, cDNA sequences and smaller engineered gene
segments that express, or may be adapted to express, proteins,
polypeptides, domains, peptides or fusion proteins.
[00106] In particular embodiments, the invention concerns DNA sequences that
encode an SP-C polypeptide that includes within its amino acid
sequence a contiguous amino acid sequence of the mouse (GenBank
accession number AAA40010; SEQ ID N0:3) or human (GenBank
accession numbers AAC32022; SEQ ID NO:S and AAC32023; SEQ
ID N0:6) SP-C and SP-Cl polypeptides, respectively, or functional
equivalents thereof.
[00107] Naturally, where the DNA segment encodes an SP-C polypeptide, or is
intended for use in expressing the SP-C polypeptide, the most preferred
sequences are those that are essentially as set forth in the contiguous
sequence of SEQ D7 NO:1, SEQ ID N0:2 or SEQ >D N0:4, and that
encode a protein that retains SP-C biological activity. Sequence of an
SP-C polypeptide will substantially correspond to a contiguous portion
of that shown in SEQ ID N0:3, SEQ ID NO:S or SEQ ID N0:6, and
have relatively few amino acids that are not identical to, or a
biologically functional equivalent of, the amino acids shown in SEQ ID
N0:3, SEQ ID NO:S or SEQ )D N0:6. The term "biologically
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functional equivalent" is well understood in the art and is further
defined in detail herein.
[0010] Accordingly, sequences that have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more
preferably, between about 91 % and about 99%; of amino acids that are
identical or functionally equivalent to the amino acids of SEQ m
N0:3, SEQ m NO:S or SEQ m N0:6 will be sequences that are
"essentially as set forth in SEQ m N0:3, SEQ m NO:S or SEQ m
N0:6.
[00109] In certain other embodiments, the invention concerns isolated DNA
segments and recombinant vectors that include within their sequence a
contiguous nucleic acid sequence from that shown in SEQ m NO:1
SEQ m N0:2, or SEQ m N0:4. This definition is used in the same
sense as described above and means that the nucleic acid sequence
substantially corresponds to a contiguous portion of that shown in SEQ
m NO:1 SEQ m N0:2, or SEQ m N0:4, and has relatively few
codons that are not identical, or functionally equivalent, to the codons
of SEQ m NO:1 SEQ ID N0:2, or SEQ m N0:4. The term
"functionally equivalent codon" is used herein to refer to codons that
encode the same amino acid and also refers to codons that encode
biologically equivalent amino acids.
[00110] Excepting intronic or flanking regions, and allowing for the
degeneracy
of the genetic code, sequences that have between about 70% and about
79%; or more preferably, between about 80% and about 89%; or even
more preferably, between about 90% and about 99% of nucleotides that
are identical to the nucleotides shown in the sequences of SEQ m
NO:1 SEQ m N0:2, or SEQ m N0:4 will be sequences that are
"essentially as set forth in SEQ B7 NO:1 SEQ m N0:2, or SEQ m
N0:4". Sequences that are essentially the same as those set forth in
SEQ m NO:1 SEQ m NO:2, or SEQ m N0:43 may also be
functionally defined as sequences that are capable of hybridizing to a
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nucleic acid segment containing the complement of SEQ ID NO:1 SEQ
ID NO:2, or SEQ ID N0:4 under relatively stringent conditions.
Suitable relatively stringent hybridization conditions will be well
known to those of skill in the art.
[00111] Naturally, the present invention also encompasses DNA segments that
are complementary, or essentially complementary, to the sequence set
forth in SEQ ID NO:1 SEQ ID N0:2, or SEQ ID NO:4. Nucleic acid
sequences that are "complementary" are those that are capable of base-
pairing according to the standard Watson-Crick complementarity rules.
As used herein, the term "complementary sequences" means nucleic
acid sequences that are substantially complementary, as may be
assessed by the same nucleotide comparison set forth above, or as
defined as being capable of hybridizing to the nucleic acid segment of
SEQ ID NO:1 or SEQ ID NO:3 under relatively stringent conditions.
[00112] It will also be understood that this invention is not limited to the
particular nucleic acid and amino acid sequences of SEQ ID NO:1,
SEQ ID N0:2, SEQ ID NO:3, SEQ ID N0:4, SEQ ID NO:S, or SEQ
ID N0:6. DNA segments may therefore variously include the SP-C
coding regions themselves, coding regions bearing selected alterations
or modifications in the basic coding region, or they may encode larger
polypeptides that nevertheless include SP-C-coding regions or may
encode biologically functional equivalent proteins or peptides that have
variant amino acids sequences.
[00113] The DNA segments of the present invention encompass biologically
functional equivalent SP-C proteins and peptides. Such sequences may
arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively, functionally
equivalent proteins or peptides may be created via the application of
recombinant DNA technology, in which changes in the protein
structure may be engineered, based on considerations of the properties
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of the amino acids being exchanged. Changes may be introduced
through the application of site-directed mutagenesis techniques, e.g., to
introduce improvements to the antigenicity of the protein or to test SP-
C mutants in order to examine adenosine deaminase activity at the
molecular level.
[00114] As modifications and changes may be made in the structure of SP-C
genes and proteins of the present invention, and still obtain molecules
having like or otherwise desirable characteristics, such biologically
functional equivalents are also encompassed within the present
invention.
[00115] For example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of interactive
binding capacity with structures such as, for example, antigen-binding
regions of antibodies, binding sites on substrate molecules or receptors,
DNA binding sites, or such like. Since it is the interactive capacity and
nature of a protein that defines that protein's biological functional
activity, certain amino acid sequence substitutions can be made in a
protein sequence (or, of course, its underlying DNA coding sequence)
and nevertheless obtain a protein with like (agonistic) properties. It is
thus contemplated by the inventors that various changes may be made
in the sequence of SP-C proteins or polypeptides, or underlying DNA,
without appreciable loss of their biological utility or activity.
[00116] In terms of functional equivalents, it is well understood by the
skilled
artisan that, inherent in the definition of a "biologically functional
equivalent protein or peptide or gene", is the concept that there is a
limit to the number of changes that may be made within a defined
portion of the molecule and still result in a molecule with an acceptable
level of equivalent biological activity. Biologically functional
equivalent peptides are thus defined herein as those peptides in which
certain, not most or all, of the amino acids may be substituted.
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[00117] In particular, where shorter length peptides are concerned, it is
contemplated that fewer amino acid substitutions should be made
within the given peptide. Longer domains may have an intermediate
number of changes. The full length protein will have the most tolerance
for a larger number of changes. Of course, a plurality of distinct
proteins/peptides with different substitutions may easily be made and
used in accordance with the invention.
[00118] Amino acid substitutions are generally based on the relative
similarity
of the amino acid side-chain substituents, for example, their
hydrophobicity, hydrophilicity, charge, size, and the like. An analysis
of the size, shape and type of the amino acid side-chain substituents
reveals that arginine, lysine and histidine are all positively charged
residues; that alanine, glycine and serine are all a similar size; and that
phenylalanine, tryptophan and tyrosine all have a generally similar
shape. Therefore, based upon these considerations, arginine, lysine and
histidine; alanine, glycine and serine; and phenylalanine, tryptophan
and tyrosine; are defined herein as biologically functional equivalents.
[00119] In one embodiment, the clone to be used in producing the knockout
construct is digested with a restriction enzyme selected to cut at a
locations) such that a marker gene can be inserted at that location in
the gene. In alternative embodiments, DNA sequences can be removed
by partial digestion with a random nuclease at a single restriction
enzyme cut in the gene.
[00120] The proper position for marker gene insertion is that which will serve
to prevent expression of the native gene. This position will depend on
various factors, including which sequences (exon, intron or promoter)
are to be targeted (i.e., the precise location of insertion necessary to
inhibit promoter function or to inhibit synthesis of the native exon) and
the availability of convenient restriction sites within the sequence. In
some cases, it is desirable to remove a large portion of the gene so as to
keep the length of the knockout construct comparable to the original
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genomic sequence when a marker gene is to be inserted into the
knockout construct.
[00121] The marker gene can be any nucleic acid sequence well known to those
skilled in the art and that is detectable and/or assayable. Typically, an
antibiotic resistance gene is used or any other gene whose expression
or presence in the genome can easily be detected. The marker gene is
usually operably linked to a promoter from any source that will be
active or can easily be activated in the cell into which it is inserted.
However, the marker gene need not have its own promoter as it may be
transcribed using the promoter of the targeted gene. In addition, the
marlcer gene will normally have a polyA sequence attached to the 3'
end of the gene for transcription termination of the gene. Preferred
marker genes are aminoglycoside phosphotransferase gene (aph), the
hygromycin B phosphotransferase gene, or any antibiotic resistance
gene known to be useful as a marker in knockout techniques.
[00122] The linear knockout construct may be transfected directly into
embryonic stern cells (discussed below), or it may first be placed into a
suitable vector for amplification prior to insertion. Suitable vectors are
known to those skilled in the art.
[00123] The invention further provides for transgenic animals, which can be
used for a variety of purposes, e.g., to identify therapeutics agents for
SP-C mediated pulmonary disorders. The transgenic animals can be
useful, e.g., for identifying drugs that modulate production of SP-C,
such as by modulating SP-C gene expression. An SP-C gene promoter
can be isolated, e.g., by screening of a genomic library with an SP-C
cDNA fragment and characterized according to methods known in the
art. In a preferred embodiment of the present invention, the transgenic
animal containing said SP-C reporter gene is used to screen a class of
bioactive molecules known as steroid hormones for their ability to
modulate SP-C expression. In a more preferred embodiment of the
invention, non-human animals are produced where the expression of
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the endogenous SP-C gene has been mutated or "knocked out". A
"knock out" animal is one carrying a homozygous or heterozygous
deletion of a particular gene or genes. These animals could be useful to
determine whether the absence of SP-C will result in a specific
phenotype, in particular whether these mice have or are likely to
develop a specific disease, such as high susceptibility to emphysema.
Furthermore these animals are useful in screens for drugs that alleviate
or attenuate the disease condition resulting from the mutation of the
SP-C gene as outlined below. In a preferred embodiment of this aspect
of the invention, a transgenic SP-C knock-out mouse, carrying the
mutated SP-C locus on both of its chromosomes, is used as a model
system for transgenic or drug treatment of the condition resulting from
loss of SP-C expression.
[00124] Methods for obtaining transgenic and knockout non-human animals are
well known in the art. In a general aspect, a transgenic animal is
produced by the integration of a given transgene into the genome in a
manner that permits the expression of the transgene, or by disrupting
the wild-type gene, leading to a knockout of the wild-type gene. U.S.
Pat. No. 5,616,491, incorporated herein by reference in its entirety,
generally describes the techniques involved in the preparation of
knockout mice.
[00125] Methods for producing transgenic animals are generally described in
U.S. Pat. Nos. 4,736,866; 4,873,191; 5,175,383; 5,824,837; 6,437,216;
6,437,215; 6,374,130, which are incorporated herein by reference in
their entirety. U.S. Pat. Nos. 5,639,457, 5,175,384; 5,175,385;
5,530,179, 5,625,125, 5,612,486 and 5,565,186 are also each
incorporated herein by reference to similarly supplement the present
teaching regarding transgenic pig, rabbit, mouse and rat production.
[00126] Knock out mice are generated by homologous integration of a "knock
out" construct into a mouse embryonic stem cell chromosome that
encodes the gene to be knocked out. In one embodiment, gene
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targeting, which is a method of using homologous recombination to
modify an animal's genome, can be used to introduce changes into
cultured embryonic stem cells. By targeting a SP-C gene of interest in
ES cells, these changes can be introduced into the germlines of animals
to generate chimeras. The gene targeting procedure is accomplished by
introducing into tissue culture cells a DNA targeting construct that
includes a segment homologous to a target SP-C locus, and which also
includes an intended sequence modification to the SP-C genomic
sequence (e.g., insertion, deletion, point mutation). The treated cells
are then screened for accurate targeting to identify and isolate those
that have been properly targeted.
[00127] Gene targeting in embryonic stem cells is in fact a scheme
contemplated by the present invention as a means for disrupting a SP-C
gene function through the use of a targeting transgene construct
designed to undergo homologous recombination with one or more SP-
C genomic sequences. The targeting construct can be arranged so that,
upon recombination with an element of a SP-C gene, a positive
selection marker is inserted into (or replaces) coding sequences of the
gene. The inserted sequence functionally disrupts the SP-C gene, while
also providing a positive selection trait. Exemplary SP-C targeting
constructs are described in more detail below.
[00128] Generally, the embryonic stem cells (ES cells) used to produce the
knockout animals will be of the same species as the knockout animal to
be generated. Thus for example, mouse embryonic stem cells will
usually be used for generation of knockout mice.
[00129] Embryonic stem cells are generated and maintained using methods well
known to the skilled artisan such as those described by Doetschman et
al. (1985) J. Embryol. Exp. Mol. Biol. 87:27-45). Any line of ES cells
can be used, however, the line chosen is typically selected for the
ability of the cells to integrate into and become part of the germ line of
a developing embryo so as to create germ line transmission of the
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knockout construct. Thus, any ES cell line that is believed to have this
capability is suitable for use herein. One mouse strain that is preferred
for production of ES cells, is the 129/Sv strain. The cells are cultured
and prepared for knockout construct insertion using methods well
known to the skilled artisan.
[00130] A typical knockout construct contains nucleic acid fragments of not
less than about 0.5 kb nor more than about 10.0 kb from both the 5'
and the 3' ends of the genomic locus which encodes the gene to be
mutated. These two fragments are separated by an intervening fragment
of nucleic acid that encodes a positive selectable marker, such as the
neomycin resistance gene (neoR). The resulting nucleic acid fragment,
consisting of a nucleic acid from the extreme 5' end of the genomic
locus linked to a nucleic acid encoding a positive selectable marker
which is in turn linked to a nucleic acid from the extreme 3' end of the
genomic locus of interest, omits most of the coding sequence for SP-C
or other gene of interest to be knocked out. When the resulting
construct recombines homologously with the chromosome at this
locus, it results in the loss of the omitted coding sequence, otherwise
known as the structural gene, from the genomic locus. A stem cell in
which such a rare homologous recombination event has taken place can
be selected for by virtue of the stable integration into the genome of the
nucleic acid of the gene encoding the positive selectable marker and
subsequent selection for cells expressing this marker gene in the
presence of an appropriate drug (neomycin in this example).
[00131] Variations on this basic technique also exist and are well known in
the
art. For example, a "knock-in" construct refers to the same basic
arrangement of a nucleic acid encoding a 5' genomic locus fragment
linked to nucleic acid encoding a positive selectable marker which in
turn is linked to a nucleic acid encoding a 3' genomic locus fragment,
but which differs in that none of the coding sequence is omitted and
thus the 5' and the 3' genomic fragments used were initially contiguous
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before being disrupted by the introduction of the nucleic acid encoding
the positive selectable marker gene. This "knock-in" type of construct
is thus very useful for the construction of mutant transgenic animals
when only a limited region of the genomic locus of the gene to be
mutated, such as a single exon, is available for cloning and genetic
manipulation. Alternatively, the "knock-in" construct can be used to
specifically eliminate a single functional domain of the targeted gene,
resulting in a transgenic animal that expresses a polypeptide of the
targeted gene that is defective in one function, while retaining the
function of other domains of the encoded polypeptide. In a variation of
the knock-in technique, a marker gene is integrated at the genomic
locus of interest such that expression of the marker gene comes under
the control of the transcriptional regulatory elements of the targeted
gene. A marker gene is one that encodes an enzyme whose activity can
be detected (e.g., ~3-galactosidase), the enzyme substrate can be added
to the cells under suitable conditions, and the enzymatic activity can be
analyzed. One skilled in the art will be familiar with other useful
marlcers and the means for detecting their presence in a given cell. For
example, one such alternative marker is the green fluorescent protein
(GFP). The GFP marker is particularly useful for the examination of
gene expression in individual viable cells. Thus GFP and related
markers are particularly useful for in situ analysis of levels of
expression of the "knocked-in" gene. All such markers are
contemplated as being included within the scope of the teaching of this
invention.
[00132] Non-homologous recombination events can be selected against by
modifying the above-mentioned knock out and knock in constructs so
that they are flanked by negative selectable markers at either end
(particularly through the use of two allelic variants of the thymidine
kinase gene, the polypeptide product of which can be selected against
in expressing cell lines in an appropriate tissue culture medium well
known in the art--i. e. one containing a drug such as 5-
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bromodeoxyuridine). Thus a preferred embodiment of such a knock out
or knock in construct of the invention consist of a nucleic acid
encoding a negative selectable marker linked to a nucleic acid encoding
a 5' end of a genomic locus linked to a nucleic acid of a positive
selectable marker which in turn is linked to a nucleic acid encoding a
3' end of the same genomic locus which in turn is linked to a second
nucleic acid encoding a negative selectable marker Nonhomologous
recombination between the resulting knock out construct and the
genome will usually result in the stable integration of one or both of
these negative selectable marker genes and hence cells which have
undergone nonhomologous recombination can be selected against by
growth in the appropriate selective media (e.g. media containing a drug
such as 5-bromodeoxyuridine for example). Simultaneous selection for
the positive selectable marker and against the negative selectable
marker will result in a vast enrichment for clones in which the knock
out construct has recombined homologously at the locus of the gene
intended to be mutated. The presence of the predicted chromosomal
alteration at the targeted gene locus in the resulting knock out stem cell
line can be confirmed by means of Southern blot analytical techniques,
which are well known to those familiar in the art. Alternatively, PCR
can be used.
[00133] Each knockout construct to be inserted into the cell must first be in
the
linear form. Therefore, if the knockout construct has been inserted into
a vector (described infra), linearization is accomplished by digesting
the DNA with a suitable restriction endonuclease selected to cut only
within the vector sequence and not within the knockout construct
sequence.
[00134] For insertion, the knockout construct is added to the ES cells under
appropriate conditions for the insertion method chosen, as is known to
the skilled artisan. For example, if the ES cells are to be electroporated,
the ES cells and knockout construct DNA are exposed to an electric
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pulse using an electroporation machine and following the
manufacturer's guidelines for use. After electroporation, the ES cells
are typically allowed to recover under suitable incubation conditions.
The cells are then screened for the presence of the knock out construct
as explained above. Where more than one construct is to be introduced
into the ES cell, each knockout construct can be introduced
simultaneously or one at a time.
[00135] After suitable ES cells containing the knockout construct in the
proper
location have been identified by the selection techniques outlined
above, the cells can be inserted into an embryo. Insertion may be
accomplished in a variety of ways known to the skilled artisan,
however a preferred method is by microinjection. For microinjection,
about 10-30 cells are collected into a micropipet and injected into
embryos that are at the proper stage of development to permit
integration of the foreign ES cell containing the knockout construct
into the developing embryo. For instance, the transformed ES cells can
be microinjected into blastocytes.
[00136] While any embryo of the right stage of development is suitable for
use,
preferred embryos are male. In mice, the preferred embryos also have
genes coding for a coat color that is different from the coat color
encoded by the ES cell genes. In this way, the offspring can be
screened easily for the presence of the knockout construct by looking
for mosaic coat color (indicating that the ES cell was incorporated into
the developing embryo). Thus, for example, if the ES cell line carries
the genes for white fur, the embryo selected will carry genes for black
or brown fur.
[00137] After the ES cell has been introduced into the embryo, the embryo may
be implanted into the uterus of a pseudopregnant foster mother for
gestation. While any foster mother may be used, the foster mother is
typically selected for her ability to breed and reproduce well, and for
her ability to care for the young. Such foster mothers are typically
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prepared by mating with vasectomized males of the same species. The
stage of the pseudopregnant foster mother is important for successful
implantation, and it is species dependent. For mice, this stage is about
2-3 days pseudopregnant.
[00138] Offspring that are born to the foster mother may be screened initially
for mosaic coat color where the coat color selection strategy (as
described above, and in the appended examples) has been employed. In
addition, or as an alternative, DNA from tail tissue of the offspring
may be screened for the presence of the knockout construct using
Southern blots and/or PCR as described above. Offspring that appear to
be mosaics may then be crossed to each other, if they are believed to
carry the knoclcout construct in their germ line, in order to generate
homozygous knockout animals. Homozygotes may be identified by
Southern blotting of equivalent amounts of genomic DNA from mice
that are the product of this cross, as well as mice that are known
heterozygotes and wild type mice.
[00139] Other means of identifying and characterizing the knockout offspring
are available. For example, Northern blots can be used to probe the
mRNA for the presence or absence of transcripts encoding either the
gene knocked out, the marker gene, or both. In addition, Western blots
can be used to assess the level of expression of the SP-C gene knocked
out in various tissues of the offspring by probing the Western blot with
an antibody against the particular SP-C protein, or an antibody against
the marker gene product, where this gene is expressed. Finally, in situ
analysis (such as fixing the cells and labeling with antibody) and/or
FACS (fluorescence activated cell sorting) analysis of various cells
from the offspring can be conducted using suitable antibodies to look
for the presence or absence of the knockout construct gene product.
[00140] Recombinase dependent knockouts can also be generated, e.g. by
homologous recombination to insert target sequences, such that tissue
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specific and/or temporal control of inactivation of a SP-C-gene can be
controlled by recombinase sequences.
[00141] Animals containing more than one knockout construct and/or more
than one transgene expression construct are prepared in any of several
ways. The preferred manner of preparation is to generate a series of
mammals, each containing one of the desired transgenic phenotypes.
Such animals are bred together through a series of crosses, backcrosses
and selections, to ultimately generate a single animal containing all
desired knockout constructs and/or expression constructs, where the
animal is otherwise congenic (genetically identical) to the wild type
except for the presence of the knockout constructs) and/or
transgene(s).
[00142] The transgenic animals of the present invention all include within a
plurality of their cells a transgene of the present invention, which
transgene alters the phenotype of the "host cell" with respect to
regulation of cell growth, death and/or differentiation. Since it is
possible to produce transgenic organisms of the invention utilizing one
or more of the transgene constructs described herein, a general
description will be given of the production of transgenic organisms by
referring generally to exogenous genetic material. This general
description can be adapted by those skilled in the art in order to
incorporate specific transgene sequences into organisms utilizing the
methods and materials described below.
[00143] In an illustrative embodiment, either the cre/loxP recombinase system
of bacteriophage P 1 or the FLP recombinase system of Saccharomyces
cerevisiae can be used to generate in vivo site-specific genetic
recombination systems, as known in the art. Cre recombinase catalyzes
the site-specific recombination of an intervening target sequence
located between loxP sequences. loxP sequences are 34 base pair
nucleotide repeat sequences to which the Cre recombinase binds and
are required for Cre recombinase mediated genetic recombination. The
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orientation of loxP sequences determines whether the intervening
target sequence is excised or inverted when Cre recombinase is
present; catalyzing the excision of the target sequence when the loxP
sequences are oriented as direct repeats and catalyzes inversion of the
target sequence when loxP sequences are oriented as inverted repeats.
[00144] Accordingly, genetic recombination of the target sequence is dependent
on expression of the Cre recombinase. Expression of the recombinase
can be regulated by promoter elements that are subject to regulatory
control, e.g., tissue-specific, developmental stage-specific, inducible or '
repressible by externally added agents. This regulated control will
result in genetic recombination of the target sequence only in cells
where recombinase expression is mediated by the promoter element.
Thus, the activation expression of a recombinant SP-C protein can be
regulated via control of recombinase expression.
[00145] Use of the crelloxP recombinase system to regulate expression of a
recombinant SP-C protein requires the construction of a transgenic
animal containing transgenes encoding both the Cre recombinase and
the subject protein. Animals containing both the Cre recombinase and a
recombinant SP-C gene can be provided through the construction of
"double" transgenic animals. A convenient method for providing such
animals is to mate two transgenic animals each containing a transgene,
e.g., a SP-C gene and recombinase gene.
[00146] In an exemplary embodiment, introducing transgenes into the germline
of the non-human animal produces the "transgenic non-human
animals" of the invention. Embryonal target cells at various
developmental stages can be used to introduce transgenes. Different
methods are used depending on the stage of development of the
embryonal target cell. The specific lines) of any animal used to
practice this invention are selected for general good health, good
embryo yields, good pronuclear visibility in the embryo, and good
reproductive fitness. In addition, the haplotype is a significant factor.
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For example, when transgenic mice are to be produced, strains such as
C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor,
Me.). Preferred strains are those with H-2b, H-2d or H-2q haplotypes
such as C57BL/6 or DBA/1. The lines) used to practice this invention
may themselves be transgenic, and/or may be knockouts (i.e., obtained
from animals that have one or more genes partially or completely
suppressed).
[00147] Z11 one embodiment, the transgene construct is introduced into a
single
stage embryo. The zygote is the best target for microinjection. In the
mouse, the male pronucleus reaches the size of approximately 20
micrometers in diameter, which allows reproducible injection of 1-2pl
of DNA solution. The use of zygotes as a target for gene transfer has a
major advantage in that in most cases the injected DNA will be
incorporated into the host gene before the first cleavage (Brinster et al.
(1985) PNAS 82:,4438-4442). As a consequence, all cells of the
transgenic animal will carry the incorporated transgene. This will in
general also be reflected in the efficient transmission of the transgene
to offspring of the founder since 50% of the germ cells will harbor the
transgene.
[00148] Normally, fertilized embryos are incubated in suitable media until the
pronuclei appear. At about this time, the nucleotide sequence
comprising the transgene is introduced into the female or male
pronucleus as described below. In some species such as mice, the male
pronucleus is preferred. It is most preferred that the exogenous genetic
material be added to the male DNA complement of the zygote prior to
its being processed by the ovum nucleus or the zygote female
pronucleus. It is thought that the ovum nucleus or female pronucleus
release molecules which affect the male DNA complement, perhaps by
replacing the protamines of the male DNA with histones, thereby
facilitating the combination of the female and male DNA complements
to form the diploid zygote.
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[00149] Thus, it is preferred that the exogenous genetic material be added to
the
male complement of DNA or any other complement of DNA prior to
its being affected by the female pronucleus. For example, the
exogenous genetic material is added to the early male pronucleus, as
soon as possible after the formation of the male pronucleus, which is
when the male and female pronuclei are well separated and both are
located close to the cell membrane. Alternatively, the exogenous
genetic material could be added to the nucleus of the sperm after it has
been induced to undergo decondensation. Sperm containing the
exogenous genetic material can then be added to the ovum or the
decondensed sperm could be added to the ovum with the transgene
constructs being added as soon as possible thereafter.
[00150] Introduction of the transgene nucleotide sequence into the embryo may
be accomplished by any means known in the art such as, for example,
microinjection, electroporation, or lipofection. Following introduction
of the transgene nucleotide sequence into the embryo, the embryo may
be incubated in vitro for varying amounts of time, or reimplanted into
the surrogate host, or both. In vitro incubation to maturity is within the
scope of this invention. One common method in to incubate the
embryos in vitro for about 1-7 days, depending on the species, and then
reimplant them into the surrogate host.
[00151] For the purposes of this invention, a zygote is essentially the
formation
of a diploid cell that is capable of developing into a complete
organism. Generally, the zygote will be comprised of an egg containing
a nucleus formed, either naturally or artificially, by the fusion of two
haploid nuclei from a gamete or gametes. Thus, the gamete nuclei must
be ones that are naturally compatible, i.e., ones that result in a viable
zygote capable of undergoing differentiation and developing into a
functioning organism. Generally, a euploid zygote is preferred. If an
aneuploid zygote is obtained, then the number of chromosomes should
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not vary by more than one with respect to the euploid number of the
organism from which either gamete originated.
[00152] In addition to similar biological considerations, physical ones also
govern the amount (e.g., volume) of exogenous genetic material, which
can be added to the nucleus of the zygote or to the genetic material,
which forms a part of the zygote nucleus. If no genetic material is
removed, then the amount of exogenous genetic material, which can be
added, is limited by the amount that will be absorbed without being
physically disruptive. Generally, the volume of exogenous genetic
material inserted will not exceed about 10 picoliters. The physical
effects of addition must not be so great as to physically destroy the
viability of the zygote. The biological limit of the number and variety
of DNA sequences will vary depending upon the particular zygote and
functions of the exogenous genetic material and will be readily
apparent to one skilled in the art, because the genetic material,
including the exogenous genetic material, of the resulting zygote must
be biologically capable of initiating and maintaining the differentiation
and development of the zygote into a functional organism.
[00153] The number of copies of the transgene constructs that are added to the
zygote is dependent upon the total amount of exogenous genetic
material added and will be the amount that enables the genetic
transfonnation to occur. Theoretically only one copy is required,
however, generally, numerous copies are utilized, for example, 1,000-
20,000 copies of the transgene construct, in order to insure that one
copy is functional. As regards the present invention, there will often be
an advantage to having more than one functioning copy of each of the
inserted exogenous DNA sequences to enhance the phenotypic
expression of the exogenous DNA sequences.
[00154] Any technique which allows for the addition of the exogenous genetic
material into nucleic genetic material can be utilized so long as it is not
destructive to the cell, nuclear membrane or other existing cellular or
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genetic structures. The exogenous genetic material is preferentially
inserted into the nucleic genetic material by microinjection.
Microinjection of cells and cellular structures is known and is used in
the art.
[00155] Reimplantation is accomplished using standard methods. Usually, the
surrogate host is anesthetized, and the embryos are inserted into the
oviduct. The number of embryos implanted into a particular host will
vary by species, but will usually be comparable to the number of off
spring the species naturally produces.
[00156] Transgenic offspring of the surrogate host may be screened for the
presence and/or expression of the transgene by any suitable method.
Screening is often accomplished by Southern blot or Northern blot
analysis, using a probe that is complementary to at least a portion of the
transgene. Western blot analysis using an antibody against the protein
encoded by the transgene may be employed as an alternative or
additional method for screening for the presence of the transgene
product. Typically, DNA is prepared from tail tissue and analyzed by
Southern analysis or PCR for the transgene. Alternatively, the tissues
or cells believed to express the transgene at the highest levels are tested
for the presence and expression of the transgene using Southern
analysis or PCR, although any tissues or cell types may be used for this
analysis.
[00157] Alternative or additional methods for evaluating the presence of the
transgene include, without limitation, suitable biochemical assays such
as enzyme and/or immunological assays, histological stains for
particular marker or enzyme activities, flow cytometric analysis, and
the like. Analysis of the blood may also be useful to detect the presence
of the transgene product in the blood, as well as to evaluate the effect
of the transgene on the levels of various types of blood cells and other
blood constituents.
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[00158] Progeny of the transgenic animals may be obtained by mating the
transgenic animal with a suitable partner, or by in vitro fertilization of
eggs and/or sperm obtained from the transgenic animal. Where mating
with a partner is to be performed, the partner may or may not be
transgenic and/or a lcnoclcout; where it is transgenic, it may contain the
same or a different transgene, or both. Alternatively, the partner may be
a parental line. Where in vitro fertilization is used, the fertilized
embryo may be implanted into a surrogate host or incubated in vitro, or
both. Using either method, the progeny may be evaluated for the
presence of the transgene using methods described above, or other
appropriate methods.
[00159] The transgenic animals produced in accordance with the present
invention will include exogenous genetic material. As set out above,
the exogenous genetic material will, in certain embodiments, be a DNA
sequence, which results in the production of a SP-C protein (either
agonistic or antagonistic), and antisense transcript, or a SP-C mutant.
Further, in such embodiments the sequence will be attached to a
transcriptional control element, e.g., a promoter, which preferably
allows the expression of the transgene product in a specific type of cell.
[00160] Retroviral infection can also be used to introduce transgene into a
non-
human animal. The developing non-human embryo can be cultured in
vitro 'to the blastocyst stage. During this time, the blastomeres can be
targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-
1264). Efficient infection of the blastomeres is obtained by enzymatic
treatment to remove the zona pellucida (Manipulating the Mouse
Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, 1986). The viral vector system used to introduce the
transgene is typically a replication-defective retrovirus carrying the
transgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et
al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently
obtained by culturing the blastomeres on a monolayer of virus-
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producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J.
6:383-388). Alternatively, infection can be performed at a later stage.
Virus or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders will be
mosaic for the transgene since incorporation occurs only in a subset of
the cells that formed the transgenic non-human animal. Further, the
founder may contain various retroviral insertions of the transgene at
different positions in the genome, which generally will segregate in the
offspring. In addition, it is also possible to introduce transgenes into
the germ line by intrauterine retroviral infection of the mid-gestation
embryo (Jahner et al. (1982) supra).
[00161] A third type of target cell for transgene introduction is the
embryonal
stem cell (ES). ES cells are obtained from pre-implantation embryos
cultured in vitro and fused with embryos. Transgenes can be efficiently
introduced into the ES cells by DNA transfection or by retrovirus-
mediated transduction. Such transformed ES cells can thereafter be
combined with blastocysts from a non-human animal. The ES cells
thereafter colonize the embryo and contribute to the germ line of the
resulting chimeric animal.
[00162] Pharmaceutical Screens
[00163] The invention provides various transgenic cell lines and organisms in
which pharmaceutical screens can be conducted to identify compounds
capable of effecting SP-C mediated pulmonary processes. As set forth
above, the transgenic cell lines and organisms are engineered to be
deficient in endogenous SP-C gene activities. The resultant loss of
endogenous SP-C activity generally leads to an "acute phase response."
The acute phase response initiates further pulmonary processes,
including those distinctive of the various pulmonary diseases and
conditions discussed above.
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[00164] Compounds identified above as being useful for preventing SP-C
mediated pulmonary processes, can be, e.g. a nucleic acid (e.g. DNA,
RNA or PNA), protein, peptide, peptidomimetic, small molecule, or
derivative thereof Preferred compounds are capable of binding to, and
regulating transcription, translation, processing, or activity of an SP-C
gene or protein. Examples include antisense, ribozyme or triplex
nucleic acids, small molecule ligands, antibody or antibody-like
binding fragments.
[00165] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., for determining the LDso (The Dose Lethal
To 50% Of The Population) and the EDSO (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be expressed as
the ratio LDSO / EDso. Compounds that exhibit large therapeutic
induces are preferred. While compounds that exhibit toxic side effects
may be used, care should be taken to design a delivery system that
targets such compounds to the site of affected tissue in order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
[00166] The data obtained from the cell culture assays and animal studies can
be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form
employed and the route of administration utilized. For any compound
used in the method of the invention, the therapeutically effective dose
can be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the ICSO (i.e., the concentration of the
test compound which achieves a half maximal inhibition of symptoms)
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as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may be
measured, for example, by high performance liquid chromatography.
[00167] Pharmaceutical compositions for use in accordance with the present
invention may be formulated in conventional manner using one or
more physiologically acceptable carriers or excipients. Thus, the
compounds and their physiologically acceptable salts and solvates may
be formulated for administration by, for example, injection, inhalation
or insufflation (either through the mouth or the nose) or oral, buccal,
parenteral or rectal administration.
(00168] For such therapy, the compounds of the invention can be formulated
for a variety of loads of administration, including systemic and topical
or localized administration. For systemic administration, injection is
preferred, including intramuscular, intravenous, intraperitoneal, and
subcutaneous. For injection, the compounds of the invention can be
formulated in liquid solutions, preferably in physiologically compatible
buffers such as Hanle's solution or Ringer's solution. In addition, the
compounds may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included.
[00169] For oral administration, the pharmaceutical compositions may take the
form of, for example, tablets or capsules prepared by conventional
means with pharmaceutically acceptable excipients such as binding
agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium
stearate, talc or silica); disintegrants (e.g., potato starch or sodium
starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The
tablets may be coated by methods well known in the art. Liquid
preparations for oral administration may take the form of, for example,
solutions, syrups or suspensions, or they may be presented as a dry
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product for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents (e.g.,
sorbitol syrup, cellulose derivatives or hydrogenated edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and
preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic
acid). The preparations may also contain buffer salts, flavoring,
coloring and sweetening agents as appropriate.
[00170] Preparations for oral administration may be suitably formulated to
give
controlled release of the active compound. For buccal administration
the compositions may take the form of tablets or lozenges formulated
in conventional manner. For administration by inhalation, the
compounds for use according to the present invention are conveniently
delivered in the form of an aerosol spray presentation from pressurized
packs or a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the
case of a pressurized aerosol the dosage unit may be determined by
providing a valve to deliver a metered amount. Capsules and cartridges
of e.g., gelatin for use in an inhaler or insufflator may be formulated
containing a powder mix of the compound and a suitable powder base
such as lactose or starch.
[00171] The compounds may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Formulations
for injection may be presented in unit dosage form, e.g., in ampoules or
in mufti-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain forinulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively, the
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active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[00172] The compounds may also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
[00173] In addition to the formulations described previously, the compounds
may also be formulated as a depot preparation. Such long acting
formulations may be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection. Thus,
for example, the compounds may be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for 'example, as a sparingly soluble salt. Other suitable
delivery systems include microspheres which offer the possibility of
local noninvasive delivery of drugs over an extended period of time,
This technology utilizes microspheres of precapillary size which can be
injected via a coronary catheter into any selected part of the e.g. heart
or other organs without causing inflammation or ischemia. The
administered therapeutic is slowly released from these microspheres
and taken up by surrounding tissue cells (e.g. endothelial cells).
[00174] Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic acid
derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be through nasal sprays
or using suppositories. For topical administration, the oligomers of the
invention are formulated into ointments, salves, gels, or creams as
generally known in the art. A wash solution can be used locally to treat
an injury or inflammation to accelerate healing.
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[00175] In situations in which the therapeutic is a gene, a gene delivery
system
can be introduced into a patient by any of a number of methods, each
of which is familiar in the art. For instance, a pharmaceutical
preparation of the gene delivery system can be introduced systemically,
e.g., by intravenous injection, and specific transduction of the protein
in the target cells occurs predominantly from specificity of transfection
provided by the gene delivery vehicle, cell-type or tissue-type
expression due to the transcriptional regulatory sequences controlling
expression of the receptor gene, or a combination thereof. In other
embodiments, initial delivery of the recombinant gene is more limited
with introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter or by
stereotactic injection. A therapeutic gene, such as a gene encoding an
antisense RNA or a ribozyme can be delivered in a gene therapy
construct by electroporation using techniques known in the art.
[00176] A gene therapy preparation can consist essentially of a gene delivery
system in an acceptable diluent, or can comprise a slow release matrix
in which the gene delivery vehicle or compound is imbedded.
Alternatively, where the complete gene delivery system can be
produced intact from recombinant cells, e.g., retroviral vectors, the
pharmaceutical preparation can comprise one or more cells which
produce the gene delivery system.
[00177) The compositions may, if desired, be presented in a pack or dispenser
device, 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.
[00178] The present invention is further illustrated by the following
examples,
which should not be construed as limiting in any way. The contents of
all cited references (including literature references, issued patents,
published patent applications as cited throughout this application are
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hereby expressly incorporated by reference. The practice of the present
invention will employ, unless otherwise indicated, conventional
techniques of cell biology, cell culture, molecular biology, transgenic
biology, microbiology, recombinant DNA, and immunology, which are
within the skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory Manual,
2nd Ed., ed. by Sambrook, Fritsch and Mauatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); '
Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B.
D. Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames ~Z S. J. Higgins eds. 1984); Culture Of Animal Cells (R.
I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes
(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning
(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,
N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and
M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In
Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical
Methods In Cell And Molecular Biology (Mayer and Walker, eds.,
Academic Press, London, 1987); Handbook Of Experimental
Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds.,
1986); Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986).
[00179]
[00180] MATERIALS AND METHODS
[00181] Animals
[00182] SP-C (-/-) mice were generated by targeted gene inactivation as
previously described [11]. Generally, a 129/J mouse ~genomic library
was screened to identify genomic clones of the SP-C gene homologous
to the 129 derived ES cells. A 2.1-kb BamHI fragment containing
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exons 2-6 of the SP-C gene was used for modification of the gene.
Sequence encoding the hydrophobic polyvaline domain of the SP-C
peptide was interrupted by insertional mutagenesis with a 1.6-kb
pGl~neo gene cassette. This insertion provided positive selection for
targeted cells by growth in the neomycin analogue 6418. The 2.1-kb
SP-C plasmid was digested with ApaLI, which cuts at a unique ApaLI
site located in the SP-C polyvaline domain. The ApaLI linker
pGI~neoBPA cassette was ligated into the SP-C ApaLI site. A 1.3-kb
PstI-to-Ba~nHI fragment spanning exon 1 and the 5' flanking DNA was
ligated to the 5' BaynHI site of the 2.1-kb Bam-pGKneoBPA fragment.
The targeting construct was modified further by cloning the herpes
simplex virus thymidine kinase gene into the 5' SphI site to provide
gancyclovir selection against nonhomologous integration of the
construct.
[00183] The D3R strain of ES cells was electroporated with the purified SP-C-
targeting construct DNA and selected as described (15). ES cell DNA
was digested with Bsu36I and analyzed with a probe outside of the
targeting construct sequence. The probe was a 457-by SphI-PstI
fragment adjacent to the 5' limit of the targeting construct. Positive
clones were confirmed by genomic Southern blot of multiple restriction
enzyme digests.
[00184] ES cell clones carrying a targeted SP-C allele were microinjected into
C57/Bl6 blastocysts and implanted into host mice. Chimeric offspring
were identified by mosaic Agouti coat color and bred to NIH Swiss
Black (Tac:N:NIHSBCfBr from Taconic Farms) females. Agouti
offspring were screened for germ-line transmission of the targeted SP-
C allele by genomic Southern blot analysis of BgIII- and SphI-digested
tail DNA. The 457 by 5' Sph-Pst fragment was used as a probe. The F1
offspring heterozygous for the SP-C mutation (+/-) were bred to
establish a colony of SP-C (+/+), SP-C (+/-), and SP-C (-/-) mice. All
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mice were maintained in a pathogen-free barrier containment facility
with filtered air, water, and autoclaved food.
[00185] Chimeric founder mice were bred to 129/Sv mice, Taconic
(Germantown, NY). Offspring were screened for transmission of the
targeted SP-C allele by genomic Southern blot analysis. Animals
positive for the targeted allele were bred to establish 129/Sv mice that
were homozygous for the targeted SP-C allele. Mice were maintained
in a barrier containment facility. All animals were handled under
aseptic condition and caged in sterilized units with filtered air, water,
and autoclaved food. Sentinel mice from this room were negative for
common viral, bacterial or parasitic pathogens. At 12 months of age,
lung homogenates prepared under aseptic conditions from SP-C (-/-)
and wild type littermates did not contain bacteria or fungus. Serology
for 23 mouse viral pathogens was negative.
[00186] Morphological Analysis
[00187] Mice were killed by intraperitoneal injection of a mixture of
ketamine,
xylazine, and acepromazine. Lungs were inflated by intratracheal
instillation of 4%. paraformaldehyde at 25 cm HZO of pressure. After
overnight fixation, the tissue was processed through conventional
paraffin embedding. Six micron tissue sections were stained with
hematoxylin-eosin, Mason's trichrome stain, or orcein stain.
Immunohistochemical staining was performed for MAC-3, MUCSA/C,
CCSP, SP-B, TTF-1, and a SMA using biotinylated primary or
secondary antibodies and avidin-biotin peroxidase (Vector Elite ABC
I~it, Vector Laboratories, Inc., Burlingame, CA) or streptavidin (Zymed
Laboratories, Burlingame, CA) using methods previously described
[13]. Electronmicroscopy was performed on lung tissue obtained from
9 month old SP-C (-l-) and age matched controls after fixation in
glutaraldehyde as previously described [14].
[00188] Phospholipid and Surfactant Proteins
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[00189] Fifteen month old mice (n=5/group) were anesthetized with
pentobarbital sodium (100 mg/kg ip) and killed by exsanguination.
Trachea was cannulated and five 1 ml aliquots of 0.9% NaCI were
flushed into the lungs and withdrawn by syringe three times for each
aliquot. The lavaged lung tissue was removed and homogenized in 2
ml of 0.9% NaCI. Saturated phosphatidylcholine (Sat PC) in lipid
extracts of bronchoalveolar lavage fluid (BALF) and lung tissue were
isolated with osmium tetroxide [15] followed by phosphorus
measurement [ 16], as previously described [ 11 ]. For phospholipid
composition analyses, extracted lipid of lung tissue after BAL were
used for two-dimensional thin-layer chromatography [17]. The spots
were visualized with iodine vapor, scraped, and assayed for phosphorus
content. Surfactant proteins in BALF were analyzed by Western blot
after SDS-PAGE [11,18].
[00190] Cytokine Measurements
[00191] Concentrations of TNF-cx, IL-113, IL-13, and IL-6 were measured in
BALF and in whole lung homogenates post-lavage. Five animals of
each genotype were assessed. ELISA kits were used according to
manufacturer's instructions (R and D Systems, Minneapolis, MN).
[00192] MMP Activity
[00193] Matrix metalloproteinase (MMP-2 and MMP-9) activity was measured
in macrophage conditioned media collected from 12 month old SP-C (-
/-) or SP-C (+/+) 129/Sv mice, as previously described [19].
Macrophages were isolated by sequential lung lavage with 1 ml of
PBS. Lavages were pooled and placed in culture at 5 x 105 cells per
well of a 24 well tissue culture dish for 24 hours in serum free RPMI
media supplemented with 1 % Nutriodoma (Boehringer Mannheim,
Indianapolis, IN) and 1% antibiotics. Proteinases from the conditioned
media were concentrated by incubation of 100 ~,l media with 15 ~,1 of
gelatin-Sepharose 4-B beads (Amersham Pharmacia, Arlington
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Heights, IL) for three hours at 4°C. The beads were pelleted by
gentle
centrifugation, washed with PBS and the proteinases eluted by
incubation of the beads in Laemmli sample buffer without BME for
one hour at 37°C. Samples were directly analyzed by electrophoresis
under nonreducing conditions into 10% Zymogram gelatin gels
(NOVEX, San Diego, CA). Gels were washed twice with 2.5% Triton
X-100 (15 minutes each) and incubated for 16 hours in a developing
buffer (50 mM Tris pH 7.5, 200 mM NaCI, 5 mM CaCl2). Gels were
then stained in 0.5% (wt/vol) Coomassie blue in 50% methanol, 10%
acetic acid followed by partial destaining to reveal the clear bands of
protease activity.
[00194] Lung Mechanics
[00195] Resistance and elastic forces were measured in airways and/or lung
parenchyma of 15 month old wild type and SP-C (-/-) mice
(n=5/group). Mice were anesthetized with 0.1 ml/10 g body weight of a
mixture (ip) containing 40 mg/ml ketamine and 2 mg/ml xylazine.
Mice were tracheostomized and respiratory impedance was measured
by using the forced oscillation technique (0.25-20 Hz) delivered by
computerized flexiVent (SCIREQ, Montreal, Canada) [20]. Estimated
total lung compliance, airway resistance, airway elastance, tissue
damping and tissue elastance for mice at 2 cm H20 PEEP were
obtained by fitting a model to each impedance spectrum. Hysteresivity
was calculated as the ratio of tissue damping to tissue elastance. With
this system, the calibration procedure removed the impedance of the
equipment and tracheal tube.
[00196] Pressure-volume relationships were studied in 10-12 month old wild
type and SP-C (-/-) mice (n=5/group). Mice were anesthetized with
pentobarbital sodium (100 mg/kg ip) and placed in a box containing
100% OZ to ensure complete collapse of the alveoli by 02 absorption.
After the mice were killed by exsanguination, the cannula was inserted
into trachea, connected to a pressure sensor (Mouse Pulmonary Testing
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System, TSS, Cincinnati, OH) and lung volume per kilogram body
weight was determined at intervals of 5 cm H20 during inflation and
deflation [21].
[00197] RESULTS
[00198] SP-C (-/-) 129/Sv Congenic Mice
[00199] SP-C (+/-) chimeric founders generated from 129/Sv embryonic stem
cells and were bred to 129/Sv mice. Since only ES cell derived sperm
transmit the SP-C mutation from the chimeric male founder, SP-C (-/-)
offspring were produced entirely from 129/Sv germ cells. Thus, the
SP-C (-/-) offspring represent an inbred strain. Poor health and reduced
fecundity were noted in SP-C (-/-) mice by 2 months of age. Few litters
were produced by animals older than 6 months of age. Poor grooming,
and conjunctivitis were noted in all SP-C (-/-) 129/Sv animals beyond
6 months of age. Deterioration of coat condition was observed in most
SP-C (-/-) mice after 2 months of age. The average body weight of 12-
13 month old SP-C (-/-) mice was reduced by 24% (25.7 g ~ 3.2, n=7
vs 33.5 g + 3.0, n=7) compared to controls. In these older SP-C (-/-)
mice, relative heart weight was increased as determined by heart/body
weight ratios. Ratios were increased by 30% with the right ventricle
being more enlarged than the left 0.00565 ~ 0.00026, m ~ SD, n=10
(SP-C -l-) vs 0.00431 + 0.00033, m ~ SD, n=7 (SP-C +l+), p<0.007.
[00200] Morphological Changes in the Lungs of SP-C (-/-) Mice
[00201] While lung structure of SP-C (-/-) mice was normal at birth (data not
shown), enlargement of alveoli was observed by 2 months of age and
thereafter, consistent with the development of emphysema, Figure 1.
Alveolar septation was irregular with absent or shortened alveolar
septal tips observed throughout the lung parenchyma. Multifocal
cellular infiltrates that generally consisted of alveolar macrophages and
other mononuclear cells were detected, Figure 1B. In lungs from 6
month old mice, consolidated parenchymal infiltrates were commonly
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observed. Extensive regions of type II cell hyperplasia and interstitial
thickening were observed in the lung parenchyma. The extent and
severity of parenchyma) abnormalities and cellular infiltrates increased
with age, often resulting in regions with complete obliteration of some
alveolar spaces at 12 months of age. Areas with epithelial cell
hyperplasia, interstitial thickening, and fibrosis were observed in
alveoli and airways. Extensive perivascular, and peribronchiolar
monocytic infiltrates were detected in the most severely affected
animals, Figure 1F.
[00202] Alveolar Remodeling
[00203] Trichrome staining demonstrated regions of fibrosis in the lung
parenchyma, pleural surfaces and at perivascular and peribronchiolar
sites, Figure 2. In some regions, collagen deposition was distributed in
extended web-like configurations throughout the lung parenchyma.
Extensive ex SMA staining, indicating myofibroblast transformation,
was observed throughout the alveoli of SP-C (-/-) mice. The intensity
and extent of a SMA staining was in general, increased with age, but
variable within lung sections and among littermates, Figure 2. Loss of
the network of alveolar elastin fibers detected with orcein stain was
observed in areas of alveolar disruption in the SP-C (-/-) mice, Figure
2. Regions with reduced orcein staining colocalized with sites of
increased trichrome staining, supporting the concept that the severity of
alveolar remodeling was correlated with pulmonary fibrosis in SP-C
deficient mice.
[00204] Electronmicroscopic Findings
[00205] At the electronmicroscopic level, alveoli of the SP-C (-/-) mice were
often thickened and lined by hyperplastic type II epithelial cells, Figure
3A. Increased numbers of cuboidal cells were observed lining alveolar
surfaces, and type II cells contained excessive numbers of lamellar
bodies. Capillary walls were thickened or obliterated by surrounding
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stroma and collagen. Bronchi and bronchioles were lined by a highly
atypical columnar epithelia. Conducting airways were lined by non-
ciliated columnar epithelial cells that contained numerous atypical
electron dense organelles, consistent with the atypical mitochondria
characteristic of Clara cells [22]. Type II cells were hypertrophic,
containing increased numbers of lamellar bodies and lipid inclusions.
In the alveolus, basement membranes were thickened, containing
numerous collagen fibrils. Many capillary lumena were obliterated, and
regions of fibrosis were readily discerned. Basement membranes and
endothelial surfaces of larger vessels were disrupted. Abnormal
alveolar macrophages contained large accumulations of surfactant like
material with structural features of tubular myelin and lamellar bodies,
Figure 3.
[00206] Macrophage Morphology and Abnormal Lipid Accumulations
[00207] Subsets of mononuclear cells in the alveolar spaces of SP-C (-/-) mice
stained intensely with the MACS antibody, an alveolar macrophage cell
marker, Figure 4B. Abnormal intracellular lipid inclusions were
observed in alveolar macrophages, Figure 4D. Likewise, lipid
accumulations were also noted in the hyperplastic type II epithelial
cells lining residual alveoli, Figure 4D. At the ultrastructural level, the
atypical alveolar macrophages contained abundant surfactant
components including lamellar bodies and tubular myelin, extracellular
forms of pulmonary surfactant, Figure 3. Other macrophages contained
numerous cytoplasmic crystals consistent with those formed by Yml, a
mammalian lectin [23]. Mass spectroscopic analysis confirmed the
presence of increased Yml in the BALF (data not shown).
Accumulation of the intracellular crystals and lipids was not detected
in alveolar macrophages from control 129/Sv maintained in this barrier
facility. In BALF from 6 month old SP-C (-/-) mice, the number of
alveolar macrophages was increased 4.4 fold, 9021 ~ 1017 vs 2039 ~
497, (n=5) in SP-C (-/-) vs SP-C (+/+), respectively. The percentage of
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lymphocytes was not altered. Changes in polymorphonuclear cells and
eosinophils were not observed.
[00208] Epithelial Cell Dysplasia
[00209] Pronounced changes in conducting airway epithelial cell morphology
were observed in SP-C (-/-) mice, Figure S. Epithelial cell dysplasia
was readily apparent at 6 to 12 months of age, the conducting airways
being lined by hyperplastic, pseudostratified columnar epithelium,
Figures 1F, SB. While MUCSA/C staining cells were rarely seen in
wild type mice, MUCSA/C positive cells lined most of the conducting
airways of the SP-C (-l-) mice, Figure SD. MUCSA/C staining of
conducting airways was generally extensive, however heterogeneity in
the pattern of staining occurred. Immunostaining for Clara cell
secretory protein (CCSP) and proSP-B was detected, but the extent and
intensity of staining was decreased in severely affected conducting
airways in SP-C (-/-) mice, also consistent with epithelial cell dysplasia
(data not shown). In the alveoli, septal thickening and dense monocytic
infiltration were noted in the areas of extensive epithelial hyperplasia.
However, in some areas with severe airspace remodeling, some alveoli
lacked type II cells. In those lesions, web-like strands of squamous
cells formed alveoli that were devoid of capillaries.
[00210] Pulmonary Mechanics
[00211] At higher pressures on the deflation limb of pressure-volume curves,
lung volumes were significantly increased in SP-C (-/-) compared to
wild type mice (Figure 6), consistent with the emphysema observed
histologically, Figure 1. At lower pressures, lung volumes were normal
and residual lung volumes were maintained at 0 pressure, consistent
with normal surfactant function. Similarly, there were no significant
differences between SP-C (-l-) and control mice in dynamic lung
compliance obtained with ventilation volumes of 7 mllkg, Table II.
While airway and tissue elastance was unaltered, both airway
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resistance and tissue damping was significantly increased in SP-C (-/-)
mice (p<0.05). Hysteresivity was significantly increased in the SP-C (-
/-) mice (p<0.01). These findings are consistent with the observed
emphysema and with maintenance of surfactant function. Surfactant
Composition Tissue and total surfactant phospholipid pool sizes were
increased approximately 2-fold in SP-C (-/-) mice, Figure 7. The
composition of lipids in lung tissue after BAL was unchanged, Table I.
SP-A, SP-B, and SP-D were estimated by Western blot analysis of
BALF. While surfactant protein B levels were unaltered, SP-A and SP-
D were significantly increased in SP-C (-/-) mice.
TABLE I. Phospholipid Content in Lung Tissue
SP-C (+/+) SP-C (-/-)
~mol/kg ~.mol/kg
SM 6.80.6 5.91.0
Lyso 8.41.0 21.53.3
PC 1
PC 70.03.1 82.512.4
PI 2.80.2 2.50.4
PS 5.31.2 6.10.6
PE 22.9 1.5 25.814.0
PG 3.30.8 4.20.5
Values are mean ~ SE, n=5 per group.
SM: ,Sphingomyelin ; PS: Phosphatidylserine ; PC:
Phosphatidylcholine ; PE: Phosphatidylethanolamine ; PI:
Phosphatidylinositol ; PG: Phosphatidylglycerol
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TABLE II. Mechanical Parameters of the Lung obtained by
using Forced Oscillation Technique
SP-C (+/+) SP-C (-/-)
Compliance (ml/cmH20~kg)1.450.11 0.690.01
Airway Resistance (cmHZO~s/ml)20.20.5 2.380.03
Airway Elastance (cmHzO19.30.7
/ml)
0.1230.006
Tissue Damping (cmHzO/ml)1.540.10 0.80~0.04*
Tissue Elastance (cmH20/ml)20.10.8 3.340.21
Hysteresivity 20.0 1.0
0.167~0.008*
Values are mean ~ SE. *p<0.05 as assessed by two tailed
Student t-test, n=5 per group.
[00212] Cytokine and Metalloproteinase Expression
[00213] Concentrations of proinflammatory cytokines were determined in
BALF and lung homogenates from 6 month old mice. TNF-c~ IL-6,
MIP-2, and IL-13 were not altered in the SP-C (-/-) mice. The
supernatants of cultured alveolar macrophages from SP-C (-/-) and
(+/+) were tested for MMP activity by SDS/PAGE zyrriography at one
year of age. Gelatinase activity was readily detectable in the
conditioned media from SP-C (-/-) macrophages but was undetectable
in media from control macrophages (SP-C +/+). Proteinase bands
migrated at approximately 72 kDa and 92 kDa, consistent with MMP-2
and MMP-9, respectively (data not shown). In addition, MMP-12
mRNA was increased 3.58 fold in lung RNA from SP-C (-/-) compared
to wild type mice. The elevated expression of MMP activity may
contribute to alveolar remodeling seen in the SP-C (-/-) mice:
[00214] DISCUSSION
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[00215] A severe pulmonary disorder characterized by emphysema, epithelial
cell dysplasia, monocytic cell infiltration, pulmonary fibrosis and
abnormal lipid accumulations, was caused by targeted deletion of the
SP-C gene in a congenic strain of SP-C (-/-)/129/Sv mice.
Heterogeneous pulmonary lesions contained 1 ) thickened, fibrotic
alveolar walls that stained for a smooth muscle actin, 2) extensive
monocytic infiltrates and increased expression of metalloproteinases,
3) regions of severe emphysema with septal thinning and degeneration
of pulmonary capillaries, 4) epithelial cell dysplasia and MUCSA/C
expression in conducting airways, and 5) accumulation of intracellular
lipids in various cell types. Pathologic findings in the SP-C (-/-) mice
were consistent with, but not identical to, those seen in lungs from
patients with various conditions termed idiopathic interstitial
pneumonitis (IIP). Thus, lack of SP-C or proSP-C can be directly
linked to the pathogenesis of interstitial lung disease in mice.
[00216] A mutation in the SP-C gene was recently associated with familial
interstitial pneumonitis that was inherited as an autosomal dominant
affect [8,9]. In a sibship with mutation c460+1G-j A, resulting in an
exon 4 deletion of the proSP-C peptide, misprocessed proSP-C
accumulated within type II epithelial cells; tissue and lung lavage
material lacked the active SP-C peptide [8]. Similarly, a single base
pair substitution (L188Q) altered subcellular localization of proSP-C in
an extended family with IIP [9]. Therefore, it has been unclear whether
the severe pulmonary disease in these patients results from the lack of
SP-C, or to abnormal accumulations of misfolded mutant SP-C or
proSP-C proteins. The present studies demonstrate that the lack of SP-
C per se, can recapitulate many of the pathologic findings consistent
with various forms of adult and childhood interstitial pneumonitis.
[00217] While the absence of proSP-C and/or SP-C caused severe lung disease
in the mouse, the molecular pathogenesis of this disorder remains
unclear. At the light microscopic level, lung structure in the SP-C (-/-)
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mice was normal at E19.5 and postnatal day 1 (data not shown).
Abnormalities seen in lung structure increased with advancing age,
suggesting that emphysema and remodeling do not arise from
abnormalities in lung morphogenesis, but from ongoing injury and
repair processes. The expression of various pro-inflammatory cytokines
that have been previously associated with emphysema and
inflammation were not altered in the SP-C (-/-) mice. There was no
change in neutrophil number, and there was no evidence of viral or
bacterial infection in SP-C (-/-) mice. These findings suggest that the
remodeling and inflammation are caused by cellular abnormalities
intrinsic to the lung, and dependent upon the functions of SP-C or
perhaps the result of selective degradation of extracellular matrix by
MMPs elaborated by the macrophages rather than to susceptibility to
pathogens: MMP-9 and MMP-2 production by alveolar macrophages,
and MMP-12 mRNA levels were increased and therefore may play a
role in the pathogenesis of the lung disease in the SP-C (-/-) mice.
Increased MMP-2, MMP-9, and MMP-12 expression was previously
associated with emphysema in SP-D gene targeted mice [24].
[00218] While pro-inflammatory cytokines were not increased in the lungs of
the SP-C (-/-) mice, the lungs were infiltrated with atypical alveolar
macrophages containing numerous lipid inclusions and Yml crystals
[23]. The numbers of the abnormal macrophages were increased 4- 5
fold compared to control. Cellular infiltration was associated with
alveolar thickening and fibrosis. The myofibroblast transformation and
collagen deposition seen at the ultrastructural level were consistent
with increased a-SMA staining seen throughout the alveolar walls of
the SP-C (-/-) mice. Paradoxically, marked epithelial cell dysplasia was
observed in conducting airways in the SP-C (-/-) mice, in spite of the
fact that proSP-C is not expressed in these cells in wild type mice.
Furthermore, high levels of expression of MUCSA/C were observed in
the conducting airways at sites in which SP-C mRNA and protein are
not normally expressed. MUCSA/C is normally expressed at low levels
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in the conducting airways of mice, but is readily induced by
inflammation or inflammatory cytokines, being increased by IL-4, IL-
13, and allergens [25 for review]. These latter findings suggest that the
lack of SP-C may influence gene expression outside the alveolus,
implying that SP-C plays a role, directly or indirectly, in the conducting
airways. However, it is unclear whether cellular abnormalities in the
conducting airways of SP-C (-/-) mice are mediated directly by SP-C
dependent signaling events or might be related to SP-C dependent
modulation of surface forces or changes in mucociliary clearance in the
absence of SP-C.
[00219] The finding that severe lung disease can be caused by either the
expression of a dominantly inherited mutant proSP-C protein or the
deletion of SP-C gene suggests several potential mechanisms by which
SP-C may contribute to the pathogenesis of lIP. In the 1TP patients
described by Nogee et al. and Thomas et al. [8,9], the mutant proSP-C
protein accumulated within type II cells, potentially creating cell injury
related to the misfolding or misprocessing of the precursor protein. In
support of this concept, Conkright et al. recently demonstrated that
expression of an SP-C mutant protein caused lethal lung dysfunction ira
vivo [26]. However, the active SP-C peptide was absent in the SP-C (-
l-) mice and in patients with IPF caused by this dominantly inherited
SP-C mutation [8]. Thus, the lack of SP-C per se may be involved in
the pathogenesis of IIP. Amin et al. recently described a sibship in
' which three individuals were severely affected by IIP, each of whom
lacked detectable expression of either proSP-C or SP-C in alveolar
lavage, in spite of the failure to find mutations in the coding region of
SP-C [27]. Whether the selective lack of proSP-C or SP-C directly
caused the disorder in these patients is unclear.
[00220] Do Abnormalities in Surfactant Function Contribute to IIP?
[00221] The present findings demonstrate that the lack of SP-C per se causes a
syndrome with features of interstitial pneumonitis in mice. Since SP-C
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enhances surface properties of phospholipids in the airspace, it is
possible that the lack of SP-C alters surfactant function in time leading
to interstitial pneumonitis. However, lung phospholipid content was
unaltered in SP-C (-/-) mice in the Swiss black strain [ 11 ], and was
increased 2-fold in SP-C (-/-) mice in 129/Sv background. Surfactant
phospholipid composition, structure of lamellar bodies and tubular
myelin were generally preserved in both strains of SP-C (-/-) mice.
Changes in lung mechanics and lung histology shown in a previous
study of 8 week old Swiss black SP-C (-/-) mice were distinct from the
present study. In SP-C (-/-) Swiss blaclc mice, there was no evidence of
inflammation or emphysema. Hysteresivity was decreased while tissue
elastance and resistance were unaltered, findings consistent with a
modest abnormality of ifz. vitro surface activities of surfactant. In
contrast, the SP-C (-/-) mice in the present study showed severe
abnormalities in airway resistance, tissue damping, and hysteresivity
was significantly increased, consistent with the extensive emphysema
[28]. Furthermore, SP-B and surfactant phospholipid pool sizes were
normal or increased, consistent with the observed preservation of
surfactant function. The modestly increased levels of SP-A and SP-D
in the SP-C (-/-) 129/Sv mice may reflect changes related to chronic
lung inflammation. Thus, there is no evidence at present that surfactant
deficiency accounts for the chronic lung disease in the SP-C (-/-)
129/Sv mice, but it remains possible that subtle differences in sheer
forces not discernable in the present studies may contribute to the
disruption of lung structure and function in the SP-C (-/-) mice. Ira vitro
studies demonstrate that various growth factors, cytokines, and sheer
stress can cause myofibroblast transformation of lung fibroblasts.
Consistent with the increased a-SMA staining observed in the present
study in mice, the extensive fibrosis and myofibroblast transformation
is often seen in humans with IIP [10]. Collagen deposition and
increased numbers of fibroblasts are also readily observed within the
alveolar walls, similar to that seen in human patients with IIP. If lack of
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SP-C contributes to the pathogenesis of the pulmonary disease, therapy
in which exogenous SP-C is administered might be considered for
patients with IIP. On the other hand, if the disorder is caused by
misrouting and abnormal accumulations of SP-C or mutant SP-C, the
addition or increased expression of normal SP-C may actually
contribute to the disorder.
[00222] Does SP-C Deficiency Cause a Lipid Storage Disease?
[00223] Surfactant lipids, lamellar bodies, and tubular myelin accumulated in
the atypical macrophages, and prominent lipid droplets were observed
in the abundant fibroblasts underlying type II cells in the lungs of SP-C
(-/-) mice. These pathologic findings suggest the possibility that the
absence of SP-C alters the catabolism of surfactant, or other cellular
constituents, creating a storage disorder. In vitf°o studies have
demonstrated that SP-C enhances surfactant lipid uptake by type II
epithelial cells, functioning in a manner distinct from that of SP-A and
SP-B, the latter serving to maintain large surfactant aggregates
associated with the epithelial surfaces [29]. Thus SP-C may have both
intracellular and extracellular roles in surfactant homeostasis.
[00224] Strain Influences the Pathologic Finding in the SP-C (-/-) Mice
[00225] The severe lung disease observed in the SP-C (-/-) mice in the 129/Sv
strain contrasts sharply with the milder abnormalities seen in SP-C (-/-)
mice when maintained in outbred Swiss black background. While the
SP-C (-/-)/Swiss black mice do not have overt abnormalities in lung
structure, these mice are susceptible to lung dysfunction when placed
in hyperoxia and reduction of surfactant protein B [12]. The strong
strain-dependent influence on the SP-C (-l-) phenotype and the
heterogeneity of pulmonary lesions that vary in severity and time and
place, are consistent with findings in patients with familial idiopathic
fibrosis caused by mutations in the SP-C gene [30]. These syndromes
are clinically and pathologically distinct from the emphysema
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associated with al-antitrypsin deficiency. In IIP, clinical and
pathologic findings vary greatly in these sibships, and multiple
histopathological diagnoses have been made within the same family.
While the nature of the SP-C mutations may influence the disorder,
marked heterogeneity in severity, age of presentation, and time of
progression of pulmonary disease characterizes this disorder,
suggesting that environmental factors or other genes strongly influence
its pathogenesis. The observed strain differences in the severity of lung
disease caused by SP-C deficiency in the SP-C (-/-) mice, suggest that
the phenotype associated with SP-C deficiency or SP-C mutations may
be strongly influenced by genetic factors. While there is no evidence
that infection complicated the interpretation of the present study, lung
dysfunction in patients with IIP is exacerbated by infection.
[00226] Implications for Diagnosis and Therapy
[00227) The present study and recent human studies [8,30] provide perhaps the
first association between gene mutations and idiopathic interstitial lung
disease. Since the absence of SP-C caused severe lung disease in the
SP-C (-/-) mice, it is also possible that deficiency of SP-C, whether
genetic or secondary to injury, may contribute to acute and chronic
lung disease. The association between mutations in SP-C with IlP in
humans, makes feasible genetic testing for the risk of the disease.
Likewise, histologic diagnosis of the various pathologies caused by
mutations in the SP-C gene can be made by immunohistochemistry.
Detection of mutant SP-C genes or the presence or absence of SP-C
from BALF may provide diagnostic insights into the role of SP-C in
patients with complex lung diseases. Finally, it is unclear whether
human DP is caused by 1) the absence of SP-C and proSP-C, 2)
misfolding and misrouting of either the SP-C proprotein or the active
SP-C peptide, or 3) altered routing, processing or degradation of other
cellular components whose homeostasis is dependent upon proSP-C
and/or SP-C. If protein misfolding in type II or other lung cells
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contributes to the pathogenesis of lung disease, the misfolding of
proteins other than SP-C may be considered in the pathogenesis of
interstitial lung disease. Clarification of cellular and molecular
mechanisms causing interstitial lung disease related to abnormalities in
SP-C may provide a conceptual basis for the development of new
therapies for IIP.
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Freeman/New York, 314-315.
23. Chang, N.C., Hung, S.L, Hwa, K.Y., Kato, L, Chen, J.E., Liu, C.H.,
and Chang, A.C. 2001. A macrophage protein, Yml, transiently
expressed during inflammation is a novel mammalian lectin. J.
Bol. Claena. 276:17497-17506.
79
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24. Wert, S.E., Yoshida, M., LeVine, A.M., Ikegami, M., Jones, T.,
Ross, G.F., Fisher, J.H., Korfhagen, T.R., and Whitsett, J.A.
2000. Increased metalloproteinase activity, oxidant production,
and emphysema in surfactant protein D gene-inactivated mice.
Proc. Natl. Acad. Sci. USA 97:5972-5977.
25. Rose, M.C., Nickola, T.J., and Voynow, J.A. 2001. Airway mucus
obstruction: mucin glycoprotiens, MUC gene regulation and
goblet cell hyperplasia. Am. .I. Respir. Cell Mol. Biol. 25:533-
537.
26. Conkright, J.J., Na, C.-L., and Weaver, T.E. 2002. Overexpression
of SP-C mature SP-C peptide causes neonatal lethality in
transgenic mice. Am. J. Respir. Cell Mol. Biol. 26:85- 90.
27. Arvin, R.S., Wert, S.E., Baughman, R.P., Tomashefski, J.F.,
Nogee, L.M., Brody, A.S., Hull, W.M., and Whitsett, J.A.
2001. Surfactant protein deficiency in familial interstitial lung
disease. J. Pediatr. 139:85-92.
28. Pillow, J.J., Korfliagen, T.R., Ikegami, M., and Sly, P.D. 2001.
Overexpression of TGFalpha increases lung tissue hysteresvity
in transgenic mice. J. Appl. Physiol. 91:2730- 2734.
29. Horowitz, A.D., Moussavian, B., and Whitsett, J.A. 1996. Roles of
SP-A, SP-B and SP-C in modulation of lipid uptake by
pulmonary epithelial cells in vitro. Am J. Physiol. (Lung Cell.
Mol. Physiol.) 270:L69-L79.
30. Nogee, L.M., Dunbar, A.E., Wert, S., Askin, F., Hamvas, A., and
Whitsett, J.A. 2002. Mutations in the surfactant protein C gene
associated with interstitial lung disease. Chest 121:205-215.
OTHER EMBODIMENTS
[00229] While the invention has been described in conjunction with the
detailed description thereof, the foregoing description is intended to
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
illustrate and not limit the scope of the invention, which is defined by
the scope of the appended claims. Other aspects, advantages, and
modifications are within the scope of the following claims.
81
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SEQUENCE LISTING
<110> Whitsett, Jeffrey A.
Glasser, Stephan W.
<120> METHODS OF DIAGNOSIS AND TREATMENT OF INTERSTITIAL LUNG
DISEASE
<130> 0010872/0507287
<150> US 60/431,949
<151> 2002-12-09
<160> 6
<170> PatentIn version 3.2
<210> 1
<211> 3633
<212> DNA
<213> Mus musculus
<300>
<308> M38314
<309> 1999-07-28
<313> (1)..(3633)
<220>
<221> gene
<222> (321)..(3443)
<220>
<221> mRNA
<222> (321) . . (3443)
<223> join (321..389, 1619..1777, 2112..2234, 2472..2582,
2911..3066,
3268..3443)
<220>
<221> exon
<222> (321) . . (389)
<220>
<221> Intron
<222> (390) . . (161.8)
<220>
<221> exon
<222> (1619)..(1777)
<220>
<1221> Intron
<222> (1778) . . (2111)
<220>
<221> exon
<222> (2112) . . (2234)
<220>
<221> Intron
<222> (2235) . . (2471)
<220>
1/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
<221> exon
<222> (2472)..(2582)
<220>
<221> Intron
<222> (2583) . . (2910)
<220>
<221> exon
<222> (2911) . . (3066)
<220>
<221> Intron
<222> (3067)..(3267)
<220>
<221> exon
<222> (3268)..(3443)
<400> 1
ctgcaggggc aggtgccagc aagagaggca gacatgcaga aagacaccca cggagagagg
agcaggtggc ccgagggtga gtttgcttga cctcacccag gtttgctctt gttggggcca
120
agaggattca tgtgcctagg ccaagggcct tgggggctct tgcagctgcc ttatcggggc
180
ctgggctctg aaaagccagg aacaaacaag ctacaaagcc aaggacttgg ctggcagaca
240
ggaggcccag tccttcaccc ctgtcctctc tgtctctgat gatatatata agacactggt
300
cacaccagag agatgaggag agg aga gag aga gag aaa cct tac aaa atg gac
353
Arg Arg Glu Arg Glu Lys Pro Tyr Lys Met Asp
1 5 10
atg agt agc aaa gag gtc ctg atg gag agt cca ccg gtgagtgtga
399
Met Ser Ser Lys Glu Val Leu Met Glu Ser Pro Pro
15 20
ttgtgtgtgt gtgtgtgtgt gctgcgcgcg catacatagt atactgactt gactgtccat
459
cctccagtag gctttttttc tttttctaga cccatatact caattagctt aggattgggc
519
ttttaggata gagataccgt gatcattaag agtccctggt agaggcaggc acgacgacca
579
cgaattcctg tataaaggga ttgagagcca cagaggggtc aatagaaagt cctgtgaaga
639
acactgatag caccaccatg tagttgggag gctacaggaa gtaccacaga gcaaatgaca
699
gtgaggctgg gatgtagatc agtagctaga gcactttgca aaacgtggag accctggggt
759
2/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
ttcagtcccc cagatccaca taatccaggc atagaggtgc acactgtttg gaaatataag
819
caagataatc attcagggtc atccttagat atataaggag ttgaaaggca gcctgggtca
879
tagatatggt ttttttcttt aaaaaaaaaa aaaggatggc tcagtaggta gtggtgcttg
939
caccaaatgt ggcggctcga gtttaatctc tggaattcac atgatgaaag gagatgaaaa
999
gttgtccctg acttacacac acacatgtac atacacactc acacatacta cacacatgta
1059
cacaaacatt cacactcaca ccacatacac acactctcac acacaccaca cacatgtgca
1119
cacacacaca cacacatgta ccttcacaca catcacacac atgcacactc aaacatacca
1179
cacaatgtat acacgctcac acactaaatc cgtgtaaaaa accatgaagc tggcacatgg
1239
actggattga gaaggaacaa aggagacccg tgtaaaggta tagagacagg gctacctgga
1299
tcctttgagg gagcaaaaac tcaggatatg tctggggtgt taggcgtcgt gggaaaacag
1359
aagaagagat cggtagctgg ctctcttgga ttatttcaaa cctagacttt gcttctaact
1419
ttctaagtag ctatggtttt gttagggtcc aaccaccttc cccaaagcct tccttcccaa
1479
aagcctctga tctccccagc tcttccttca cactcgtagc ctaaagaggt acgggatgtg
1539
tgtgtccccc ttgcacaggg agagtaggag ccaccctctc cactacccac ttgtctctct
1599
gcttcccttg ctctctcag gat tac tcg gca ggt ccc agg agc cag ttc cgc
1651
Asp Tyr Ser Ala Gly Pro Arg Ser Gln Phe Arg
25 30
atc ccc tgc tgt ccc gtg cac ctc aaa cgc ctt ctc atc gtg gtt gtg
1699
Ile Pro Cys Cys Pro Val His Leu Lys Arg Leu Leu Ile Val Val Val
35 40 45 50
gtg gtg gtc ctc gtt gtc gtg gtg att gta ggg get ctg ctc atg ggc
1747
Val Val Val Leu Val Val Val Val Ile Val Gly Ala Leu Leu Met Gly
55 60 65
ctc cac atg agt caa aaa cat act gag atg gtgagtgggc ctgggttggg
1797
Leu His Met Ser Gln Lys His Thr~Glu Met
70 75
caaagaggca cagcagacag ggggttgggg gagattatgg gggatgggca gctgttcgga
1857
3/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
ggaagagaag ggagtggaca ggtatgagca catcttgggt gacacaaaca gagacgaggt
1917
agccatcctg cctagatcct ctcccccagc cccggcctag tgtgataacc atcgattgct
1977
ctgacacctc ttacgtttag ccttcctgag atctcaggaa agcgtttgaa tagaggattc
2037
tgagtagata tgggtaccga aagctgaggg aagaaagaga aataccaggc agcattccaa
2097
cacccttccc ctag gtc ctt gag atg agc atc gga gca ccg gaa act cag
2147
Val Leu Glu Met Ser Ile Gly Ala Pro Glu Thr Gln
80 85
aaa cgc cta gcc ccg agt gag cga gca gac acc atc get acc ttt tcc
2195
Lys Arg Leu Ala Pro Ser Glu Arg Ala Asp Thr Ile Ala Thr Phe Ser
90 95 100
atc ggc tcc act ggc atc gtt gtg tat gac tac cag cgg gtgaggatgc
2244
Ile Gly Ser Thr Gly Ile Val Val Tyr Asp Tyr Gln Arg
105 110 115
cggaggacca ccgggacttt attggaacta gccagttgta gcatttctag aggtctctcc
2304
ccattctgtg cctggctacc tcacctcaga tgctcgaacc actgacgcaa gtgcgcccct
2364
ccaccctctg aagacaatct aaaggaagtt ggttggctga gaactagggt tggggaggaa
2424
gcaaggcaag gggaccttgt gaatgacctc cagggtttta tacctag ctc ctg acg
2480
Leu Leu Thr
120
gcc tat aag cca get cca gga acc tac tgc tac atc atg'aag atg get
2528
Ala Tyr Lys Pro Ala Pro Gly Thr Tyr Cys Tyr Ile Met Lys Met Ala
125 130 135
cca gag agc atc cct agt ctt gag get ttc get aga aaa ctc cag aac
2576
Pro Glu Ser Ile Pro Ser Leu Glu Ala Phe Ala Arg Lys Leu Gln Asn
140 145 150
ttc agg tgggtatgtt tagggaggga gggagcagtc tcctctgagg tttgagtaga
2632
Phe Arg
agggacatgt gaaagatgac tagcgtaccc tgtgtagtat tgatgtttct gatcagacat
2692
gttctcctct ctccatgacc tgtgtcctgc catccctacc agctctcagg tggccctgct
2752
4/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
aagttgttgg ccttggctga gcttagacat gacctatggg cttctacatc caacccagtc
2812
cctctctgaa tttgtgagga aactgatcct cgagaattac caacttagtg tcccacacta
2872
aataaagcag gtgacattga aagtaggtgt tctttcca gcc aag ccc tcc aca ccc
2928
Ala Lys Pro Ser Thr Pro
155 160
acc tct aag ctg ggc cag gag gaa ggg cat gat act ggt tcc gag tcc
2976
Thr Ser Lys Leu Gly Gln Glu Glu Gly His Asp Thr Gly Ser Glu Ser
165 170 175
gat tct tcc ggg aga gac ctg get ttc cta ggc ctt get gtg agc acc
3024
Asp Ser Ser Gly Arg Asp Leu Ala Phe Leu Gly Leu Ala Val Ser Thr
180 185 190
ctg tgt gga gag cta cca ctc tac tat atc tag cat cca cag
3066
Leu Cys Gly Glu Leu Pro Leu Tyr Tyr Ile His Pro Gln
195 200 205
gtgagcaaca gtacctttca gggtgcctgg gcaacactgg cagggcttgg gctgcctgct
3126
ttgtcagggg acctactagg tatctcttaa agtcagtggt ctcgggagct cggaggatgg
3186
agggttccca gacacatccc acactggacc caagcgggtg gcttcttcag tccccataag
3246
cattagttct ttgcttcaca g ggt cgg tag aaa ccg cag cgg gac agg aaa
3297
Gly Arg Lys Pro Gln Arg Asp Arg Lys
210
gac cct ccg caa agg gtc ttt gtc aga caa gca gga agc tgc tcc tgc
3345
Asp.Pro Pro Gln Arg Val Phe Val Arg Gln Ala Gly Ser Cys Ser Cys
215 220 225 230
cca gaa acc ggt gga agt ctg taa agg aaa ggt gtc tct cct acg ggc
3393
Pro Glu Thr Gly Gly Ser Leu Arg Lys Gly Val Ser Pro Thr Gly
235 240 245
cag ggg gat cct atc aca aaa gaa taa agc agc ctg att gga aaa caa
3441
Gln Gly Asp Pro Ile Thr Lys Glu Ser Ser Leu Ile Gly Lys Gln
250 255 260
ag agtggcgctt cttttctttc acattttctc agctcggctt ctagcagaag
3493
ctcctaggaa ggagagggtt tggagagttg ggcgctttgc acgtcctttc taagagtgat
3553
ggaggtttgg tctaccccag gtaatgggaa cagagcaaaa ggcagggaca gaggacggga
3613
5/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
cctgggtgcc atacacgggg
3633
<210> 2
<211> 995
<212> DNA
<213> Mus musculus
<400> 2
aggagagaga gagagaaacc ttacaaaatg gacatgagta gcaaagaggt cctgatggag
agtccaccgg attactcggc aggtcccagg agccagttcc gcatccectg ctgtcccgtg
120
cacctcaaac gccttctcat cgtggttgtg gtggtggtcc tcgttgtcgt ggtgattgta
180
ggggctctgc tcatgggcct ccacatgagt caaaaacata ctgagatggt ccttgagatg
240
agcatcggag caccggaaac tcagaaacgc ctagccccga gtgagcgagc agacaccatc
300
gctacctttt ccatcggctc cactggcatc gttgtgtatg actaccagcg gctcctgacg
360
gcctataagc cagctccagg aacctactgc tacatcatga agatggctcc agagagcatc
420
cctagtcttg aggctttcgc tagaaaactc cagaacttca gggccaagcc ctccacaccc
480
acctctaagc tgggccagga ggaagggcat gatactggtt ccgagtccga ttcttccggg
540
agagacctgg ctttcctagg ccttgctgtg agcaccctgt gtggagagct accactctac
600
tatatctagc atccacaggt gagcaacagt acctttcagg'gtgcctgggc aacactggca
660
gggcttgggc tgcctgcttt gtcaggggac ctactaggta tctcttaaag tcagtggtct
720
cgggagctcg gaggatggag ggttcccaga cacatcccac actggaccca agcgggtggc
780
ttcttcagtc cccataagca ttagttcttt gcttcacagg gtcggtagaa accgcagcgg
840
gacaggaaag accctccgca aagggtcttt gtcagacaag caggaagctg ctcctgccca
900
gaaaccggtg gaagtctgta aaggaaaggt gtctctccta cgggccaggg ggatcctatc
960
acaaaagaat aaagcagcct gattggaaaa caaag
995
<210> 3
<211> 193
6/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
<212> PRT
<213> Mus musculus
<300>
<308> AAA40010
<309> 1999-07-28
<313> (1) . . (193)
<400> 3
Met Asp Met Ser Ser Lys Glu Val Leu Met Glu Ser Pro Pro Asp Tyr
1 5 10 15
Sex Ala Gly Pro Arg Ser Gln Phe Arg Ile Pro Cys Cys Pro Val His
20 25 30
Leu Lys Arg Leu Leu Ile Val Val Val Val Val Val Leu Va1 Val Val
35 40 45
Val Ile Val Gly Ala Leu Leu Met Gly Leu His Met Ser Gln Lys His
50 55 60
Thr Glu Met Val Leu Glu Met Ser Ile Gly Ala Pro Glu Thr Gln Lys
65 70 75 80
Arg Leu Ala Pro Ser Glu Arg Ala Asp Thr Ile Ala Thr Phe Ser Ile
85 90 95
Gly Ser Thr Gly Ile Val Val Tyr Asp Tyr Gln Arg Leu Leu Thr Ala
100 105 110
Tyr Lys Pro Ala Pro Gly Thr Tyr Cys Tyr Ile Met Lys Met Ala Pro
115 120 125
Glu Ser Ile Pro Ser Leu Glu Ala Phe Ala Arg Lys Leu Gln Asn Phe
130 135 140
Arg Ala Lys Pro Ser Thr Pro Thr Ser Lys Leu Gly Gln Glu Glu Gly
145 150 155 160
His Asp Thr Gly Ser Glu Ser Asp Ser Ser Gly Arg Asp Leu Ala Phe
165 170 175
Leu Gly Leu Ala Val Ser Thr Leu Cys Gly Glu Leu Pro Leu Tyr Tyr
180 185 190
Ile
<210> 4
<211> 3409
7/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
<212>DNA
<213>Homo Sapiens
<300>
<308>J03890
<309>1998-08-17
<313>(1) . . (3409)
<220>
<221> gene .
<222> (591) . . (3237)
<220>
<221> mRNA
<222> (591) .. (3237)
<223> join(616..657,1356..1514,1860..1982,2206..2316,2651..2809);
gene="SP-C1"; product="pulmonary surfactant protein SP-C"
<220>
<221> mRNA
<222> (591)..(3237)
<223> join(616..657,1356..1514,1860..1982,2206..2316,2669..2809);
gene="SP-C1"; product="pulmonary surfactant protein SP-C1"
<220>
<221> exon
<222> (591)..(657)
<220>
<221> Intron
<222> (658)..(1355)
<220>
<221> exon
<222> (1356)..(1514)
<220>
<221> Intron
<222> (1515)..(1859)
<220>
<221> exon
<222> (1860)..(1982)
<220>
<221> Intron
<222> (1983)..(2205)
<220>
<221> exon
<222> (2206)..(2316)
<220>
<221> Intron
<222> (2317)..(2650)
<220>
<221> exon
<222> (2651)..(3237)
<400> 4
ggtaccagat atgtgggagg aggcaaggta agggaaagag tacttgaagt tggaactggt
8/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
ccttgcaggg aaatgcacat ttatgaaacc ccgaaaactg atgtcaaagc acctcctgcc
120
ttgggcagtc ctctcagagt ctacaggtgc tgcctccaga accctcttcc tggagcgcat
180
ccctatgtat ctagaaattc tgctgggaaa tatgatggtc agacccttgg ccacctgaaa
240
gttcagggtg gtagaagaaa aaggaaagcc acagggcagc aggggcaggt gcagcaagga
300
aggcaggcac gccaggaaga cacccatggg tagaagtgca gatggcccga gggcacagtt
360
tgctcaactc acccaggttt gctcttgctg gggccaagag gactcatgtg ccagggccaa
420
gggctctggg ggctctcaca gggggcttat ctgggcttcg gttctggagg gccaggaaca
480
aacaggcttc aaagcaaggg cttggctggc acacaggggc ttggtccttc acctctgtcc
540
ctctcctacg gacacatata agaccctggt cacacctggg agaggaggag agg aga
596
Arg Arg
1
gca tag cac ctg cag caa gat gga tgt ggg cag caa aga ggt cct gat
644
Ala His Leu Gln Gln Asp Gly Cys Gly Gln Gln Arg Gly Pro Asp
10 15
gga gag ccc gcc g gtgagtgtgg ttgcgtgtgt gtatgtatgt gcgcgcgcac
697
Gly Glu Pro Ala
atgtgtgtga tggccctgcc tcctctatcc tccctggcct gtttccttat ccagatccat
757
tcactcaact aacctaggac tgtgataagt caggatgggg acaccaagac cactaagcca
817
gggacccttg gggagctgtt tgtggccaag agccactata ggggtccgta gaactggagt
877
gcgcgtagac agccctgagt cagaagccat gagaaacttc agaagtcagg ggacacttct
937
cagagaaaaa ccacatacga gctggagcca gaataaggag gagctcgccc ggtggagaag
997
gaggaaggca ttccaggaag gagggagact ctgtatcacc gcatggaggt gatcacttgg
1057
ggagagagag gggctgacca tggctggggg aagcagcagg gagagacagg tgaagcaggc
1117
tctcttgggt ccctcaaaac tagaccctgc ttctaagctt ctatgtatct atgggtttgt
1177
9/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
tagaatccag gccacctcct ccaagaagcc ttctctgatc tcctcagccc ttccctgtcc
1237
atccatcgca tcggctgtcc agcctaggag ccgtgggagg gtgttcagct tgtataggga
1297
gaagagggga cagcctcatg acctcatgcc tgtctccttg cctgccccac cgtgtcag
1355
ga cta ctc cgc agc tcc ccg ggg ccg att tgg cat tcc ctg ctg ccc
1402
Gly Leu Leu Arg Ser Ser Pro Gly Pro Ile Trp His Ser Leu Leu Pro
25 30 35
agt gca cct gaa acg cct tct tat cgt ggt ggt ggt ggt ggt cct cat
1450
Ser Ala Pro Glu Thr Pro Ser Tyr Arg Gly Gly Gly Gly Gly Pro His
40 45 50
cgt cgt ggt gat tgt ggg agc cct get cat ggg tct cca cat gag cca
1498
Arg Arg Gly Asp Cys Gly Ser Pro Ala His Gly Ser Pro His Glu Pro
55 60 65
gaa aca cac gga gat g gtgagaggtg tgggatgcac agcagtgggc acaggacatg
1554
Glu Thr His Gly Asp
ccagacagag gggctaggtg ggatgggcga taggaaactg tccaagggga gtggagggga
1614
ggaggcaagg ggcacagcta gaaggaaaga ggcacgaacc aggcagcaac ccagctcagg
1674
cttttccaca aggcccctgc ccgcgacagg acagccagct ccctccagca cctggttcca
1734
ctcagcctcc ctgaactctt gggaaagagg gaagcgcatt tgagtacaga ggcctgagta
1794
tggggatggg taccactggc tgagtaggaa aggggaagac caggtggctc catgcctttc
1854
cccag gt tct gga gat gag cat tgg ggc gcc gga agc cca gca acg cct
1903
Gly Ser Gly Asp Glu His Trp Gly Ala Gly Ser Pro Ala Thr Pro
85
ggc cct gag tga gca cct ggt tac cac tgc cac ctt ctc cat cgg ctc
1951
Gly Pro Glu Ala Pro Gly Tyr His Cys His Leu Leu His Arg Leu
95 100
cac tgg cct cgt ggt gta tga cta cca gca g gtgggtatgc cagacctcct
2002
His Trp Pro Arg Gly Val Leu Pro Ala
105 110
gacctggacc aatgacaact gggctctgct agagcgccca gctggccact ttcattccac
2062
atccatctct cctctctcag actttttgct gagcccagat tctagtagtc tcccgtgccc
2122
10/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
aacctagagg gaggtggcta aggacctggg tcagggagag agcagggcag gaccccgaat
2182
gatctccagc attctgtgcc tag ct get gat cgc cta caa gcc agc ccc tgg
2234
Ala Ala Asp Arg Leu Gln Ala Ser Pro Trp
115 120
cac ctg ctg cta cat cat gaa gat agc tcc aga gag cat ccc cag tct
2282
His Leu Leu Leu His His Glu Asp Ser Ser Arg Glu His Pro Gln Ser
125 130 135
tga ggc tct cac tag aaa agt cca caa ctt cca g gtgtgtgtgt
2326
Gly Ser His Lys Ser Pro Gln Leu Pro
140 145
gtgggtgaaa agagtgggct gtctccctcc caggctgctg gaggagtgtc cgaatggtgg
2386
ctatttgtca cctgtaaagc actgttcctc attggctgcc agctgactgc ccctctccta
2446
ttcccctgca cgactccttt ccttcccacc ccactgccaa gctgctgggc tcagctgagt
2506
ccactcacta cctggtggct tctgactcta gcacagcccc tctttactga tgagaaaact
2566 '
gaggctcaga gagattgcct gatatacctg aagtcccaca ataagggctg cacatgggat
2626
agaaactcac ttcctacatt ccag at gga atg ctc tct gca ggc caa gcc
2676
Asp Gly Met Leu Ser Ala Gly Gln Ala
150 155
cgc agt gcc tac gtc taa get ggg cca ggc aga ggg gcg aga tgc agg
2724
Arg Ser Ala Tyr Val Ala Gly Pro Gly Arg Gly Ala Arg Cys Arg
160 165 170
ctc agc.acc ctc cgg agg gga ccc ggc ctt cct ggg cat ggc cgt gaa
2772
Leu Ser Thr Leu Arg Arg Gly Pro Gly Leu Pro Gly His Gly Arg Glu
175 180 185
cac cct gtg tgg cga ggt gcc get cta cta cat cta gga cgc ctc cgg
2820
His Pro Val Trp Arg Gly Ala Ala Leu Leu His Leu Gly Arg Leu Arg
190 195 200
tga gca ggt gtg atc cca ggg ccc ctg atc agc agc gga gga gcg ctg
2868
Ala Gly Val Ile Pro Gly Pro Leu Ile Ser Ser Gly Gly Ala Leu
205 210 215
gcc acc tgc ccg get gtg gag gag get cgc tga cca ggc tgg ggc gtc
2916
Ala Thr Cys Pro Ala Val.Glu Glu Ala Arg Pro Gly Trp Gly Val
220 225 230
11/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
cac tga agc ggg gtc atc cag gca act cgg ggg agg gga agc tca cag
2964
His Ser Gly Val Ile Gln Ala Thr Arg Gly Arg Gly Ser Ser Gln
235 240 245
acc ggt act tcc cac tcc cct gaa ttc tct ctg tcc atc ctc aac att
3 012
Thr Gly Thr Ser His Ser Pro Glu Phe Ser Leu Ser Ile Leu Asn Ile
250 255 260 265
cct ttg ctt cat agg gtc agt gga agc ccc aac gga aag gaa acg ccc
3060
Pro Leu Leu His Arg Val Ser Gly Ser Pro Asn Gly Lys Glu Thr Pro
270 275 280
cgg gca aag ggt ctt ttg cag ctt ttg cag acg ggc aag aag ctg ctt
3108
Arg Ala Lys Gly Leu Leu Gln Leu Leu Gln Thr Gly Lys Lys Leu Leu
285 290 295
ctg ccc aca ccg cag gga caa acc ctg gag aaa tgg gag ctt ggg gag
3156
Leu Pro Thr Pro Gln Gly Gln Thr Leu Glu Lys Trp Glu Leu Gly Glu
300 305 310
agg atg gga gtg ggc aga ggt ggc acc cag ggg ccc ggg aac tcc tgc
3204
Arg Met Gly Val Gly Arg Gly Gly Thr Gln Gly Pro Gly Asn Ser Cys
315 320 325
cac aac aga ata aag cag cct gat ttg aaa agc aaagggtctg cttctgtctt
3257
His Asn Arg Ile Lys Gln Pro Asp Leu Lys Ser
330 335 340
cctgcagggc gcagtcctcg ctggcggggc cggccaagaa gggaagggcc ttgggagagc
3317
aaagtggggt ttccattcgc cctctgtccc agggcgctgg cactgtccac ctcggcgggg
3377 ,
agaggggctc gcagggagca tccacgggct tt
3409
<210> 5
<211> 197
<212> PRT
<213> Homo Sapiens
<300>
<308> AAC32022
<309> 1998-08-17
<313> (1) . . (197)
<400> 5
Met Asp Val Gly Ser Lys Glu Val Leu Met Glu Ser Pro Pro Asp Tyr
1 5 10 15
Ser Ala Ala Pro Arg Gly Arg Phe Gly Ile Pro Cys,Cys Pro Val His
20 25 30
12/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
Leu Lys Arg Leu Leu Ile Val Val Val Val Val Val Leu Ile Val Val
35 40 45
Val Ile Val Gly Ala Leu Leu Met Gly Leu His Met Ser Gln Lys His
50 55 60
Thr Glu Met Val Leu Glu Met Ser Ile Gly Ala Pro Glu Ala Gln Gln
65 70 75 80
Arg Leu Ala Leu Ser Glu His Leu Val Thr Thr Ala Thr Phe Ser Ile
85 90 95
Gly Ser Thr Gly Leu Val Val Tyr Asp Tyr Gln Gln Leu Leu Ile Ala
100 105 110
Tyr Lys Pro Ala Pro G1y Thr Cys Cys Tyr Ile Met Lys Ile Ala Pro
115 120 125
Glu Ser Ile Pro Ser Leu Glu Ala Leu Thr Arg Lys Val His Asn Phe
13 0 13 5 14 0
Gln Met Glu Cys Ser Leu Gln Ala Lys Pro Ala Val Pro Thr Ser Lys
145 150 155 160
Leu Gly Gln Ala Glu Gly Arg Asp Ala Gly Ser Ala Pro Ser Gly Gly
165 170 175
Asp Pro Ala Phe Leu Gly Met Ala Val Asn Thr Leu Cys Gly Glu Val
180 185 190
Pro Leu Tyr Tyr Ile
195
<210> 6
<211> 191
<212> PRT
<213> Homo Sapiens
<300>
<308> AAC320023
<309> 1998-08-17
<313> (1) . . (191)
<400> 6
Met Asp Val Gly Ser Lys Glu Val Leu Met Glu Ser Pro Pro Asp Tyr
1 5 10 15
Ser Ala Ala Pro Arg Gly Arg Phe Gly Ile Pro Cys Cys Pro Val His
20 25 30
13/14
CA 02507613 2005-05-27
WO 2004/056310 PCT/US2003/038915
Leu Lys Arg Leu Leu Ile Val Val Val Val Val Val Leu Ile Val Val
35 40 45
Val Ile Val Gly Ala Leu Leu Met Gly Leu His Met Ser Gln Lys His
50 55 60
Thr Glu Met Val Leu Glu Met Ser Ile Gly Ala Pro Glu Ala Gln Gln
65 70 75 80
Arg Leu Ala Leu Ser Glu His Leu Val Thr Thr Al,a Thr Phe Ser Ile
85 90 95
Gly Ser Thr Gly Leu Val Val Tyr Asp Tyr Gln Gln Leu Leu Ile Ala
100 105 110
Tyr Lys Pro Ala Pro Gly Thr Cys Cys Tyr Ile Met Lys Ile Ala Pro
115 120 125
Glu Ser Ile Pro Ser Leu Glu Ala Leu Thr Arg Lys Val His Asn Phe
130 135 140
Gln Ala Lys Pro A1a Val Pro Thr Ser Lys Leu Gly Gln Ala Glu Gly
145 150 155 160
Arg Asp Ala Gly Ser Ala Pro Ser Gly Gly Asp Pro Ala Phe Leu Gly
165 l70 175
Met Ala Val Asn Thr Leu Cys Gly Glu Val Pro Leu Tyr Tyr Ile
180 185 190
14/14
