Note: Descriptions are shown in the official language in which they were submitted.
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Use Of Mullerian Inhibiting Substance For
Treating Excess Androgen States
Background of the Invention
Statement as to Rights to Inventions Made Under
Federally-Sponsored Research and Development
Part of the work performed during development of this invention utilized
U. S. Government funds from National Cancer Institute grant no. R29CA79459,
and from National Institute of Child Health and Human Development grant nos.
RO1-HD-32112, P30-HD-28138, F32-HD-07954, and U54HD31398. The U.S.
Government has certain rights in this invention.
Field of the Invention
This application is directed generally to methods of treating prostate
cancer, polycystic ovarian disease, benign prostatic hypertrophy, and
precocious
puberty.
Related Art
Mullerian inhibiting substance (MIS) is a member of the transforming
growth factor-~3 (TGF~3) family of growth and differentiation factors. After
the
sexually indifferent gonad commits to testis development under the influence
of
the testis-determining factor, SRY, Sertoli cells of the fetal testis begin
procuring
MIS, which is a phenotypic hallmark of testis development (Swain et al.,
Nature
391:761-767 (1998)). MIS, also known as anti-Mullerian hormone (Jossa et al.,
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Recent Prog. Horm. Res. 48:1-59 (1993)), is absolutely required for normal
male
reproductive tract development because it affects the regression of the
Mullerian
duct of the bipotential urogenital ridge, which, is left undisturbed, would
give rise
to female reproductive tract structures such as the uterus, Fallopian tubes,
and
upper vagina (Jost, A., Arch. Anat. Microsc. Morphol. Exp. 36:271-315 (1947);
Cate et al., Cell 45:685-698 (1986); Teixeira and Donohoe, J Androl. 17:336-
341 (1996)). Adult ovaries and testes also produce MIS, albeit at much lower
levels than in fetal males, and the functional roles played by MI5 in these
settings
have not been fully elucidated (Ueno et al., Endocrinology 123:1652-1659
(1988); Tsafriri etal., Biol. Reprod 38:481-485 (1988)). However, studies in
the
rat suggest a role for MIS in oocyte maturation (Takahashi et al., Mol. Cell
Endocrinol. 47:225-234 (1986)) and in human ovary in blocking granulosa cell
proliferation and reducing steroidogenesis (Kim et al., J. Clin. Endocrinol.
Metab.
75:911-917 (1992); Seiferetal., J. Clin. Endocrinol. Metab. 76:711-714
(1993)).
Summary of the Invention
The present invention provides a method of treating a condition or disease
characterized by an excess of one or more androgens, the method comprising
administering an effective amount of MIS to a patient.
The present invention also provides a method of treating a condition or
disease characterized by an excess of one or more androgens, the method
comprising administering an effective amount of a nucleic acid encoding MI5 to
a patient.
The present invention also provides a method of decreasing the plasma
level of one or more androgens, the method comprising administering to a
patient
an effective amount of MIS, wherein the amount of MIS is sufficient to
decrease
the plasma level of the one or more androgens below the normal level for the
one
or more androgens.
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The present invention also provides a method of decreasing the plasma
level of one or more androgens, the method comprising administering to a
patient
an effective amount of nucleic acid encoding MIS, wherein the amount of MIS is
sufficient to decrease the plasma level of the one or more androgens below the
normal level for the one or more androgens.
The methods of the present invention are particularly well-suited for the
treatment of prostate cancer, polycystic ovarian disease, benign prostatic
hypertrophy, and precocious puberty.
Brief Description of the Figures
FIG. 1 depicts the pathways of testosterone biosynthesis. 21-Carbon
testosterone is synthesized from the 27-carbon cholesterol molecule by the
activities of P450-scc (cytochrome P450 side-chain cleavage), P450c17
(cytochrome P450c 17 hydroxylase/lyase), 3 ~3HSD, and 17KSR ( 17-ketosteroid
reductase).
FIG. 2 depicts results of a study of MIS type II receptor mRNA
expression in rodent Leydig cells. Total RNA was isolated from the indicated
sources, denatured, electrophoresed, bloated to a nylon membrane, and probed
with a radio-labeled MIS type II receptor riboprobe. The blot was exposed to
x-ray film to detect the migration of hybridized mRNA. The mol. wt. marker
shown indicates the migration of the 2-kb band of a 7~HindIII digest.
FIGS. 3A and 3B depict the results of an analysis of steroid production in
MA-10 cells. Culture medium was collected and assayed by RLA for total
accumulated progesterone (FIG. 3A) and testosterone (FIG. 3B); shown are
levels
after 1 or 2 days of treatment with 1 OS nM MIS in presence or absence of 50
~M
cAMP. Cross morphology was indistinguishable between MIS-treated and
untreated cells. There was no significant difference between total protein
content
in MIS-treated and untreated cells as measured previously (Bradford, M.M.,
Anal
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Biochem. 72:248-254 (1976)). Error bars represent the SEM. *, P < 0.05; **,
P < 0.001; ***, P < 0.0001. Significance was measured using Student's t test.
FIGS. 4A-C depict a Northern analysis of the expression of RNAs for
steroidogenic enzymes. Northern analysis of the steady state levels of mRNAs
from the indicated cells for steroidogenic enzymes was performed as described
in
Materials and Methods with the indicated probes. Blots were reprobed with a
human (3-actin to control for sample loading. In FIG. 4A, R2C cells were
treated
for 3 h with MIS (105 nM) or vehicle control as indicated. Total RNA was
extracted from the cell cultures after incubation, and Northern blots were
performed using 10 ug of each sample, with 30-day postnatal rat testis RNA as
a control. In FIG. 4B, MA-10 cells were treated for 18 h with MIS (105 nM) or
vehicle control in the presence or absence of (Bu)Z-cAMP (50 ,uM) as
indicated.
In FIG. 4C, MA-10 cells were treated with 10 ~cM/ml cyclohexamide 30 min.
before treatment with MIS or vehicle control, as indicated.
FIGS. SA-C depict the results of a study of MIS regulation of the
P450c17a promoter. A promoter/reported mini-gene system employing the
luciferase reporter gene was used to characterize the promoter of the P450c17a
gene. In FIG. 5A, MA-10 cells were incubated, 24 h post-transfection, with
vehicle control or 1.4, 7, 35, 70, 105 and 175 nM MIS for 18 or 29 h. Cell
extracts were assayed for firefly luciferase and renilla luciferase activity,
and the
results are shown as firefly/renilla values normalized to vehicle control
values at
1000 relative light units. In FIG. 5B, Cells were incubated with vehicle
control
or 105 nM MIS for the indicated periods of time starting 24 h after
transfection.
Cells were also incubated with an inactive L9 noncleavable mutant MIS for 18 h
(shown with an asterisk). Cell extracts were assayed for luciferase activity,
and
the results are shown as firefly/renilla values normalized to the 18 h vehicle
control
values at 1000 relative light units. In FIG. SC, Cells were treated with 105
nM
MIS at the start of transfection and incubated for the indicated periods of
time.
Cell extracts were assayed for luciferase activity, and the results are shown
as
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firefly/renilla values normalized to 18 h vehicle control values at 1000
relative light
units. Error bars represent the SEM.
FIG. 6 depicts the results of a study of MIS regulation of testosterone
concentration iv vivo. Adult male rats (250 g) were administered either hCG
(50
U, n=3, white bar), or 50 U hCG and 1 mg MIS (n=3, black bar)
intraperitoneally.
After 18 hours, serum was collected from each of the animals and tested for
testosterone concentration using a radioimmunoassay. The mean values in each
group are shown, and error bars represent SEM, p<Ø05.
Detailed Description
The terms "mullerian inhibiting substance," "MIS," and anti-Mullerian
hormone" are synonymous.
Conditions or diseases characterized by an excess of one or more
androgens include, but are not limited to, a condition or disease selected
from the
group consisting of polycystic ovarian disease or syndrome, precocious
puberty,
and McCune-Albright syndrome. Whether androgen levels are excessive can be
readily diagnosed by one of ordinary skill in the art, using clinical
chemisty,
including urinalysis, and blood, serum, or plasma analysis.
The term "androgen" includes testosterone and its metabolites, such as
dihydrotestosterone, androsterone, estradiol, and etiocholanolone. Thus, a
condition or disease characterized by an excess of one or more androgens, such
as testosterone or one of its metabolites, is increased, relative to the
normal level
of the androgen. For example, the normal plasma level of testosterone is about
10-35 nmol/L (about 3-10 ng/mL). See Harrison's Principles of Internal
Medicine, 14"' Edition, Fauci, A. S. et al., Eds., McGaw-Hill, New York (
1998),
at page 2040, Table 332-2, the content of which is incorporated by reference.
Whether a patient has a condition or disease characterized by an excess of
one or more androgens, and particularly polycystic ovarian disease or syndrome
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or precocious puberty, can be readily diagnosed by one of ordinary skill in
the art,
based upon physical examination, clinical chemistry analysis, family history,
and
patient interviews. See Harrison's Principles of Internal Medicine, 14"'
Edition,
Fauci, A.S. et al., Eds., McGaw-Hill, New York (1998).
In patients with benign prostatic hypertrophy, to whom MIS, or nucleic
acid encoding MIS, is administered, the extent of prostatic hypertrophy is
diminished or decreased. The extent of prostatic hypertrophy, and changes
therein, can be readily determined by one of ordinary skill in the art.
In patients with prostatic cancer, to whom MIS, or nucleic acid encoding
MIS, is administered, the progression ofthe tumor, whether primary or
metastatic,
is halted or slowed. The progression of a tumor, whether primary or
metastatic,
can be readily determined by one of ordinary skill in the art, such as by
tissue
biopsy, ultrasound, magnetic resonance imaging (MRI), computer aided
tomography (CAT scan), or palpation.
Miillerian Inhibiting Substance (MIS) is produced by the fetal testis as a
140 kDa glycosylated disulfide-linked homodimer that causes regression of the
Mullerian duct in the male fetus. Under reducing conditions, the protein
migrates
on gel electrophoresis at an apparent molecular weight of 70 kDa. The protein
can be proteolytically cleaved by exogenous plasmin into two distinct
fragments
that migrate electrophoretically as 57 kDa and 12.5 kDa moieties with cleavage
at residue 427 of the intact 535 amino acid monomer (Pepinsky, et al., J.
Biol.
Chem. 263:18961-4 (1988)).
The term "carboxy-terminal (C-terminal) fragment of MIS" is intended to
include compounds and materials structurally similar to the about 12. 5 kDa
(about
25 kDa under non-reducing conditions) C-terminal fragment of MIS resulting
from proteolytic (e.g., plasmin) cleavage at residue 427 of the intact 535
amino
acid human MIS monomer. The proteolytic (e.g., plasmin) cleavage site is at
residue 443 ofthe 551 amino acid bovine MIS molecule. In particular, "carboxy
terminal (C-terminal) fragment of MIS" is intended to include the about 25 kDa
homodimeric C-terminal fragment of MIS.
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By "N-terminal fragment of MIS" is intended the about 57 kDa fragment
resulting from the above-noted cleavage at residue 427 of the intact 535 amino
acid human MI5 monomer (residue 443 of the 551 amino acid bovine MIS).
The complete sequence nucleotide sequence for MIS is disclosed in U.S.
Patent No. 5,047,336, which is hereby incorporated by reference. The C-
terminal
amino acid and nucleotide sequences for bovine MIS are shown in Figure 17 of
U. S. Patent No. 5,661,126, which is hereby incorporated by reference in its
entirety. The C-terminal amino acid and nucleotide sequences for human MIS are
shown in Figure 18 of U. S. Patent No. 5,661,126. A comparison of the amino
acid sequence for human and bovine MIS, showing the - and C-terminal domains
is shown in Cate et al., Handbook of Experimental Pharmacology 95/11:184,
edited by M.B. Spoon and A.B. Roberts, Spinger-Verlag Berlin Heidelberg
( 1990), which are hereby incorporated by reference.
Additionally, the methods of the present invention can be practiced using
mutant forms of the C-terminal fragment of MIS which have substantially the
same biological activity as the C-terminal fragment of MI5. Examples of such
mutant forms would be C-terminal fragment ofMIS molecules carrying a deletion,
insertion, or alteration of amino acid sequence. In particular, the C-terminal
fragment of MIS can be modified to increase its half life in vivo. For
example,
addition of one or more amino acids or other chemical agents to the amino
and/or
carboxyl end of the C-terminal fragment can be used to increase the fragment's
stability.
The C-terminal fragment of MI5 can be obtained from a mammalian
source or through the use of recombinant DNA technology, or from chemical
synthesis of the C-terminal polypeptide.
A gene is said to be a "recombinant" gene if it results from the application
of Recombinant DNA Techniques. Examples of recombinant DNA techniques
include cloning, mutagenesis, transformation, etc. Recombinant DNA Techniques
are disclosed in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y. (1982, 1989). "Recombinant MIS" refers to MIS
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polypeptide, or a fragment thereof, and particularly the C-terminal fragment,
that
is prepared using recombinant means.
Recombinant MIS can be expressed in a protein expression system. The
use of prokaryotic and eukaryotic expression systems is well understood by
those
of ordinary skill in the art. For example, bacterial (e.g., E. coli), fungi
(e.g.,
yeast), mammalian cells (e.g., CHO cells, COS cells) or insect cells (e.g.,
baculovirus cells) expression systems can be used. For example, the C-terminal
fragment (human or bovine) can be readily produced by the recombinant DNA
techniques described in U. S. Patent No. 5,047,336, which is fully
incorporated by
reference herein. Of particular interest is expression of the C-terminal
fragment
in E. coli and other bacteria, since the C-terminal fragment is not
glycosylated.
Within a specific cloning or expression vehicle, various sites may be
selected for insertion of the gene coding for MIS or C-terminal fragment of
MIS.
These sites are usually designated by the restriction endonuclease which cuts
them
and are well recognized by those of skill in the art. Various methods for
inserting
DNA sequences into these sites to form recombinant DNA molecules are also well
known. These include, for example, dG-dC or dA-dT tailing, direct ligation,
synthetic linkers, exonuclease and polymerase-linked repair reactions followed
by
ligation, or extension of the DNA strand with DNA polymerase and an
appropriate single-stranded template followed by ligation. It is, of course,
to be
understood that a cloning or expression vehicle useful in this invention need
not
have a restriction endonuclease site for insertion of the chosen DNA fragment.
Instead, the vehicle could be joined to the fragment by alternative means.
Various expression control sequences may also be chosen to effect the
expression of recombinant DNA sequences. These expression control sequences
include, for example, the lac system, the ~3-lactamase system, the t~ system,
the
tac system, the trc system, the major operator and promoter regions of phase
~,,
the control regions of fd coat protein, the promoter for 3-phosphoglycerate
kinase
or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS,
the
promoters of the yeast a-mating factors, promoters for mammalian cells such as
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the S V40 early promoter, adenovirus late promoter and metallothionine
promoter,
and other sequences known to control the expression of genes of prokaryotic or
eukaryotic cells or their viruses and various combinations thereof. In
mammalian
cells, it is additionally possible to amplify the expression units by linking
the gene
to that for dihydrofolate reductase and applying a selection to host Chinese
hamster ovary cells.
For expression of recombinant DNA sequences, these DNA sequences are
operatively-linked to one or more of the above-described expression control
sequences in the expression vector. Such operative linking, which may be
effected
before or after the MIS or C-terminal fragment of MIS DNA sequence is inserted
into a cloning vehicle, enables the expression control sequences to control
and
promote the expression of the DNA sequence.
The vector or expression vehicle, and in particular the sites chosen therein
for insertion of the selected DNA fragment and the expression control sequence
employed in this invention, is determined by a variety of factors, e.g.,
number of
sites susceptible to a particular restriction enzyme, size of the protein to
be
expressed, expression characteristics such as start and stop codons relative
to the
vector sequences, and other factors recognized by those of skill in the art.
The
choice of a vector, expression control sequence, and insertion site for the
MIS or
C-terminal fragment of MIS DNA sequence is determined by a balance of these
factors, not all selections being equally effective for a given case.
It should also be understood that the DNA sequences coding for MIS or
the C-terminal fragment of MIS that are inserted at the selected site of a
cloning
or expression vehicle may include nucleotides which are not part of the actual
gene coding for MIS or the C-terminal fragment of MIS or may include only a
fragment of the actual gene. It is only required that whatever DNA sequence is
employed, a transformed host will produce MIS or the C-terminal fragment of
MIS. For example, the MIS DNA sequences ofthis invention may be fused in the
same reading frame in an expression vector of this invention to at least a
portion
of a DNA sequence coding for at least one eukaryotic or prokaryotic signal
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sequence, or combinations thereof. Such constructions enable the production
of,
for example, a methionyl or other peptidyl-MIS polypeptide, that is part of
this
invention. This N-terminal methionine or peptide may either then be cleaved
intra-
or extra-cellularly by a variety of known processes or the MIS polypeptide
with
the methionine or peptide attached may be used, uncleaved, in the
pharmaceutical
compositions and methods of this invention.
The cloning vehicle or expression vector containing the MIS or C-terminal
fragment of MIS polypeptide coding sequences of this invention is employed in
accordance with this invention to transform tumor cells so as to permit
expression
of an effective amount of MIS or an effective amount of the C-terminal
fragment
of MIS to inhibit primary or metastatic tumor growth.
As indicated, it should be understood that the MIS polypeptide (prepared
in accordance with this invention) may include polypeptides in the form of
fused
proteins (e.g., linked to prokaryotic, eukaryotic or combination N-terminal
segment to direct excretion, improve stability, improve purification or
improve
possible cleavage at amino acid residue 443 to release an active C-terminal
fragment), in the form of a precursor of MIS (e.g., starting with all or parts
of a
MIS signal sequence of other eukaryotic or prokaryotic signal sequences), in
the
form of a mature MIS polypeptide, or in the form of an fmet-MIS polypeptide.
The present invention also encompasses substituting codons for those of
the MIS or C-terminal fragment ofMIS nucleotide sequences. These substituted
codons may code for amino acids identical to those coded for by the codons
replaced but result in higher yield of the polypeptide. Alternatively, the
replacement of one or a combination of codons leading to amino acid
replacement
or to a longer or shorter polypeptide may alter its properties in a useful way
(e.g.,
increase the stability, increase the solubility or increase the therapeutic
activity).
Alternatively, non-recombinant MIS or a fragment thereof, and particularly
the C-terminal fragment, can be used in the methods of the present invention.
Methods for purifying non-recombinant MIS are well-known to those of ordinary
skill in the art. See U.S. Patent Nos. 4,404,188, 4,487,833 and 5,011,687.
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MIS Polypeptide Delivery Methods
MIS polypeptide, or a fragment thereof, and MIS-encoding nucleic acid
can be administered in the form of a pharmaceutical compositions. The
pharmaceutical composition can be formulated according to known methods to
prepare pharmaceutically useful compositions, whereby MIS or the C-terminal
fragment of MIS or their functional derivatives are combined in admixture with
a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their
formulation, inclusive of other human proteins, i.e., human serum albumin, are
described for example in Remington's Pharmaceutical Sciences, 18th edition, A.
R. Gennaro, Ed., Mack Publ., Easton, PA (1990). In order to from a
pharmaceutically acceptable composition suitable for effective administration,
such
compositions will contain an effective amount of MIS or the C-terminal
fragment
of MIS, or their functional derivatives, together with a suitable amount of
carrier
vehicle.
In one embodiment ofthe present invention, an "effective amount" ofMIS
is one which is sufficient to inhibit the progression of and/or reduce the
severity
of polycystic ovarian disease or syndrome, or to slow and/or halt the
progression
of precocious puberty. Likewise, an "effective amount" of the C-terminal
fragment of MIS is one which is sufficient to inhibit the progression of
and/or
reduce the severity of polycystic ovarian disease or syndrome, or to slow
and/or
halt the progression of precocious puberty.
In another embodiment of the present invention, an "effective amount" of
MIS is one which is sufficient to decrease the plasma level of one or more
androgens, including testosterone and/or its metabolites, such as
dihydrotestosterone, androsterone, estradiol, and etiocholanolone, below the
normal level. The normal plasma level oftestosterone below about 10-35 nmol/L
(about 3-10 ng/mL). See Harrison's Principles of Internal Medicine, 14'"
Edition, Fauci, A.S. et al., Eds., McGaw-Hill, New York (1998), at page 2040,
Table 332-2, the content of which is incorporated by reference. In a preferred
embodiment, an effective amount ofMIS is one which is sufficient to decrease
the
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plasma level of testosterone below about 10-35 nmol/L (about 3-10 ng/mL). In
a particularly preferred embodiment, an effective amount of MIS is one which
is
sufficient to decrease the plasma level of testosterone below about 15-30
nmol/L.
In a still more preferred embodiment, an effective amount of MIS is one which
is
sufficient to decrease the plasma level oftestosterone below about 20-25
nmol/L.
The effective amount may vary depending upon criteria such as the age,
weight, physical condition, past medical history, and sensitivity of the
recipient.
The effective amount will also vary depending on whether administration is
oral,
intravenous, intramuscular, subcutaneous, local, or by direct application to
the
tumor. In the case of direct tumor application, it is preferable that a final
serum
concentration of at least 0.1 nM, preferably about 0.1-1.0 nM, of MIS be
achieved. Likewise, for direct tumor application of the C-terminal fragment of
MIS, it is preferable that a final serum concentration of at least 0.1 nM,
preferably
about 0.1-1.0 nM, of the C-terminal fragment of MIS be achieved. Effective
individual dosage through the additionally named means of administration can
be
readily determined by methods well known to those of ordinary skill in the
art.
For example, using the size ratio calculation as detailed above, one of
ordinary
skill in the art can determine optimal dosage levels for any means of
administration. In treating a patient, it is preferable to achieve a serum
level of at
least 10 ng/ml of MIS. In treating a patient with the C-terminal fragment of
MIS,
it is preferable to achieve a serum level ranging from about 1 ng/ml to about
20
gg/ml of the C-terminal fragment of MIS.
A composition is said to be "pharmacologically acceptable" if its
administration can be tolerated by a recipient patient. Such an agent is said
to be
physiologically significant if its presence results in a detectable change in
the
physiology of a recipient patient.
Compositions containing MIS or the C-terminal fragment of MIS or their
functional derivatives may be administered orally, intravenously,
intramuscularly,
subcutaneously, or locally. Additional pharmaceutical methods may be employed
to control the duration of action. Controlled release preparations may be
achieved
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by the use of polymers to complex or adsorb MIS or the C-terminal fragment of
MIS or their functional derivatives. The controlled delivery may be exercised
by
selecting appropriate macromolecules (for example polyesters, polyamino acids,
polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose,
carboxymethylcellulose, and protamine sulfate) and the concentration of
macromolecules as well as the methods of incorporation in order to control
release.
Another possible method to control the duration of action by controlled
release preparations is to incorporate MIS or the C-terminal fragment of MIS
into
particles of a polymeric material such as polyesters, polyamino acids,
hydrogels,
poly(lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead
of
incorporating MIS or the C-terminal fragment of MIS into these polymeric
particles, it is possible to entrap MIS or the C-terminal fragment of MIS in
microcapsules prepared, for example, by coacervation techniques or by
interfacial
polymerization, for example, hydroxymethylcellulose or gelatin microcapsules
and
poly(methylmethacrylate) microcapsules, respectively, or in colloidal drug
delivery
systems, for example, liposomes, albumin microspheres, microemulsions,
nanoparticles, and nanocapsules or in macroemulsions. Such teachings are
disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro,
Ed., Mack Publ., Easton, PA (1990).
A "functional derivative" of MIS is a compound which possesses a
biological activity (either functional or structural) that is substantially
similar to a
biological activity ofMIS. The term "functional derivatives" is intended to
include
the "fragments," "variants," "analogs," or "chemical derivatives" of a
molecule.
A "fragment" of a molecule such as either MIS, is meant to refer to any
polypeptide subset of the molecule. Fragments of MIS which has activity and
which are soluble (i.e not membrane bound) are especially preferred. A
"variant"
of a molecule such MIS is meant to refer to a molecule substantially similar
in
structure and function to either the entire molecule, or to a fragment
thereof. A
molecule is said to be "substantially similar" to another molecule if both
molecules
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have substantially similar structures or if both molecules possess a similar
biological activity. Thus, provided that two molecules possess a similar
activity,
they are considered variants as that term is used herein even if the structure
of one
of the molecules not found in the other, or if the sequence of amino acid
residues
is not identical. An "analog" of a molecule such as MIS is meant to refer to a
molecule substantially similar in function to either the entire molecule or to
a
fragment thereof. As used herein, a molecule is said to be a "chemical
derivative"
of another molecule when it contains additional chemical moieties not normally
a
part of the molecule. Such moieties may improve the molecule's solubility,
absorption, biological half life, etc. The moieties may alternatively decrease
the
toxicity of the molecule, eliminate or attenuate any undesirable side effect
of the
molecule, etc. Moieties capable of mediating such effects are disclosed in
Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack
Publ., Easton, PA (1990). "Toxin-derivatized" molecules constitute a special
class of"chemical derivatives." A "toxin-derivatized" molecule is a molecule
(such
as MIS or an antibody to its receptor) which contains a toxin moiety. The
binding
of such a molecule to a cell brings the toxin moiety into close proximity with
the
cell and thereby promotes cell death. Any suitable toxin moiety may be
employed;
however, it is preferable to employ toxins such as, for example, the ricin
toxin, the
diphtheria toxin, radioisotopic toxins, membrane-channel-forming toxins, etc.
Procedures for coupling such moieties to a molecule are well known in the art.
MIS (or its functional derivatives, agonists, or antagonists) may be
administered to patients intravenously, intramuscularly, subcutaneously,
enterally,
or parenterally. When administering such compounds by injection, the
administration may be by continuous infusion, or by single or multiple
boluses.
MIS molecules (and their functional derivatives, agonists and antagonists
can be formulated according to known methods to prepare pharmaceutically
useful compositions, whereby these materials, or their functional derivatives,
are
combined in admixture with a pharmaceutically acceptable carrier vehicle.
Suitable vehicles and their formulation, inclusive of other human proteins,
e.g.,
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human serum albumin, are described, for example, inRemington'sPharmaceutical
Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publ., Easton, PA (1990). In
order to form a pharmaceutically acceptable composition suitable for effective
administration, such compositions will contain an effective amount of MIS
molecule, or its functional derivatives, agonists, or antagonists, together
with a
suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the
duration of action. Control release preparations may be achieved through the
use
of polymers to complex or absorb MIS or its functional derivatives, agonists,
or
antagonists. The controlled delivery may be exercised by selecting appropriate
macromolecules (for example polyesters, polyamino acids, polyvinyl,
pyrrolidone,
ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine,
sulfate) and the concentration of macromolecules as well as the methods of
incorporation in order to control release. Another possible method to control
the
duration of action by controlled release preparations is to incorporate the
MIS
molecule, or its functional derivatives, agonists, or antagonists, into
particles of
a polymeric material such as polyesters, polyamino acids, hydrogels,
poly(lactic
acid) or ethylene vinylacetate copolymers. Alternatively, instead of
incorporating
these agents into polymeric particles, it is possible to entrap these
materials in
microcapsules prepared, for example, by coacervation techniques or by
interfacial
polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules
and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug
delivery systems, for example, liposomes, albumin microspheres,
microemulsions,
nanoparticles, and nanocapsules or in macroemulsions. Such techniques are
disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro,
Ed., Mack Publ., Easton, PA (1990). The compositions of the present invention
may be prepared as articles of manufacture, such as "kits." Preferably, such
kits
will contain two or more containers which are specially adapted to receive MIS
or one of its functional derivatives, and an agonist of MIS.
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The term "patient" is intended to include animal patients. More preferably,
"patient" is intended to include mammalian patients, most preferably, human
patients.
The term "protein fragment" is meant to include both synthetic and
naturally-occurring amino acid sequences derivable from the naturally
occurring
amino acid sequence of MIS. The protein is said to be "derivable from the
naturally-occurring amino acid sequence of MIS" if it can be obtained by
fragmenting the naturally-occurring chosen sequence of MIS, or if it can be
synthesized based upon a knowledge of the sequence of the naturally occurring
amino acid sequence or ofthe genetic material (DNA or RNA) which encodes this
sequence.
MLS' Gene Therapy Methods
In one embodiment of the present invention, an "effective amount" of
nucleic acid encoding MIS is one which is su~cient to inhibit the progression
of
and/or reduce the severity of polycystic ovarian disease or syndrome, or to
slow
and/or halt the progression of precocious puberty. Likewise, an "effective
amount" of nucleic acid encoding the C-terminal fragment of MIS is one which
is
sufficient to inhibit the progression of and/or reduce the severity of
polycystic
ovarian disease or syndrome, or to slow and/or halt the progression of
precocious
puberty.
In another embodiment of the present invention, an "effective amount" of
nucleic acid encoding MIS is one which is sufficient to decrease the plasma
level
of one or more androgens. including testosterone and/or its metabolites, such
as
dihydrotestosterone, androsterone, estradiol, and etiocholanolone, below the
normal level. The normal plasma level of testosterone below about 10-3 5
nmol/L
(about 3-10 ng/mL). See Harrison :sPrinciples o, f'Internal Medicine, 14'''
Edition,
Fauci, A. S. et al., Eds., McGaw-Hill, New York ( 1998), at page 2040, Table
332-
2, the content of which is incorporated by reference. In a preferred
embodiment,
an effective amount of MIS is one which is sufficient to decrease the plasma
level
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oftestosterone below about 10-35 nmol/L (about 3-10 ng/mL). In a particularly
preferred embodiment, an effective amount of MIS is one which is sufficient to
decrease the plasma level of testosterone below about 15-30 nmol/L. In a still
more preferred embodiment, an effective amount ofMIS is one which is
sufficient
to decrease the plasma level of testosterone below about 20-25 nmol/L,.
Whether a vector contains a gene capable of expressing an "effective
amount of MIS" or an "effective amount of the C-terminal fragment of MIS" can
be determined following the protocols set forth in Example 4 in U.S. Patent
No.
5,661,126, which is hereby incorporated by reference in its entirety.
The gene therapy methods relate to the introduction of nucleic acid (DNA,
RNA and antisense DNA or RNA) sequences into an animal to achieve expression
of the MIS polypeptide of the present invention. This method requires a
polynucleotide which codes for an MI5 polypeptide operatively linked to a
promoter and any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery techniques
are
known in the art, see, for example, W090/11092, which is herein incorporated
by
reference.
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) comprising a promoter operably linked to an MI5
polynucleotide ex vivo, with the engineered cells then being provided to a
patient
to be treated with the polypeptide. Such methods are well-known in the art.
For
example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85:207-216 (1993);
Ferrantini, M. etal., Cancer Research 53:1107-1112 (1993); Ferrantini, M.
etal.,
J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);
Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L.,
et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene
Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one
embodiment, the cells which are engineered are arterial cells. The arterial
cells
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may be reintroduced into the patient through direct injection to the artery,
the
tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the MI5 polynucleotide constructs can
be delivered by any method that delivers injectable materials to the cells of
an
animal, such as, injection into the interstitial space oftissues (heart,
muscle, skin,
lung, liver, and the like). The MIS polynucleotide constructs may be delivered
in
a pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, the MIS polynucleotide is delivered free of any
delivery vehicle that acts to assist, promote or facilitate entry into the
cell. In
another embodiment, the MIS polynucleotide is delivered free of viral
sequences.
In another embodiment, the MIS polynucleotide is delivered free ofviral
particles.
In another embodiment, the MIS polynucleotide is delivered free of liposome
formulations. In another embodiment, the MIS polynucleotide is delivered free
of lipofectin. In another embodiment, the MI5 polynucleotide is delivered free
of
precipitating agents. However, the MI5 polynucleotides can also be delivered
in
liposome formulations and lipofectin formulations and the like can be prepared
by
methods well known to those skilled in the art. Such methods are described,
for
example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are
herein incorporated by reference.
The MI5 polynucleotide vector constructs used in the gene therapy
method are preferably constructs that will not integrate into the host genome
nor
will they contain sequences that allow for replication. Appropriate vectors
include
pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene;
pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI/V5,
pcDNA3 .1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will
be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for
driving the expression of MIS DNA. Suitable promoters include adenoviral
promoters, such as the adenoviral major late promoter; or heterologous
promoters, such as the cytomegalovirus (CMV) promoter; the respiratory
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syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter,
the metallothionein promoter; heat shock promoters; the albumin promoter; the
ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such
as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin
promoter; and human growth hormone promoters. The promoter also may be the
native promoter for MIS.
Unlike other gene therapy techniques, one major advantage ofintroducing
naked nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have shown that non-replicating
DNA sequences can be introduced into cells to provide production of the
desired
polypeptide for periods of up to six months.
The MIS polynucleotide construct can be delivered to the interstitial space
of tissues within the an animal, including of muscle, skin, brain, lung,
liver, spleen,
bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,
gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye,
gland, and connective tissue. Interstitial space of the tissues comprises the
intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of
organ
tissues, elastic fibers in the walls of vessels or chambers, collagen fibers
of fibrous
tissues, or that same matrix within connective tissue ensheathing muscle cells
or in
the lacunae of bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid ofthe lymphatic channels. Delivery to the
interstitial
space of muscle tissue is preferred for the reasons discussed below. They may
be
conveniently delivered by injection into the tissues comprising these cells.
They are
preferably delivered to and expressed in persistent, non-dividing cells which
are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such as, for
example, stem
cells of blood or skin fibroblasts. In vivo muscle cells are particularly
competent
in their ability to take up and express polynucleotides.
For the naked acid sequence injection, an effective dosage amount of DNA
or RNA will be in the range of from about 0.05 mg/kg body weight to about 50
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mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to
about
20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of
course, as the artisan of ordinary skill will appreciate, this dosage will
vary
according to the tissue site of injection. The appropriate and effective
dosage of
nucleic acid sequence can readily be determined by those of ordinary skill in
the art
and may depend on the condition being treated and the route of administration.
The preferred route of administration is by the parenteral route ofinjection
into the interstitial space of tissues. However, other parenteral routes may
also
be used, such as, inhalation of an aerosol formulation particularly for
delivery to
lungs or bronchial tissues, throat or mucous membranes of the nose. In
addition,
naked MIS DNA constructs can be delivered to arteries during angioplasty by
the
catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous injection, topical administration, catheter infusion, and so-
called "gene
guns". These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents,
etc. Such methods of delivery are known in the art.
In certain embodiments, the MIS polynucleotide constructs are complexed
in a liposome preparation. Liposomal preparations for use in the instant
invention
include cationic (positively charged), anionic (negatively charged) and
neutral
preparations. However, cationic liposomes are particularly preferred because a
tight charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al. , Proc. Natl. Acad. Sci.
USA
( 1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone
et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein
incorporated by reference); and purified transcription factors (Debs et al. ,
J. Biol.
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Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in
functional form.
Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly useful and are available under the trademark Lipofectin, from
GIBCO
BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
(1987) 84:7413-7416, which is herein incorporated by reference). Other
commercially available liposomes include transfectace (DDAB/DOPE) and
DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials
using techniques well known in the art. See, e.g. PCT Publication No. WO
90/11092 (which is herein incorporated by reference) for a description of the
synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)
liposomes. Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is
herein
incorporated by reference. Similar methods can be used to prepare liposomes
from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from
Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using
readily
available materials. Such materials include phosphatidyl, choline,
cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine
(DOPE), among others. These materials can also be mixed with the DOTMA and
DOTAP starting materials in appropriate ratios. Methods for making liposomes
using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can be used in various combinations to make conventional liposomes,
with or without the addition of cholesterol. Thus, for example, DOPG/DOPC
vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a
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stream of nitrogen gas into a sonication vial. The sample is placed under a
vacuum
pump overnight and is hydrated the following day with deionized water. The
sample is then sonicated for 2 hours in a capped vial, using a Heat Systems
model
350 sonicator equipped with an inverted cup (bath type) probe at the maximum
setting while the bath is circulated at 15EC. Alternatively, negatively
charged
vesicles can be prepared without sonication to produce multilamellar vesicles
or
by extrusion through nucleopore membranes to produce unilamellar vesicles of
discrete size. Other methods are known and available to those of skill in the
art.
The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs
being preferred. The various liposome-nucleic acid complexes are prepared
using
methods well known in the art. See, e.g., Straubinger et al., Methods of
Immunology ( 1983 ), 101: 512-527, which is herein incorporated by reference.
For
example, MLVs containing nucleic acid can be prepared by depositing a thin
film
of phospholipid on the walls of a glass tube and subsequently hydrating with a
solution of the material to be encapsulated. SUVs are prepared by extended
sonication of MLVs to produce a homogeneous population of unilamellar
liposomes. The material to be entrapped is added to a suspension of preformed
MLVs and then sonicated. When using liposomes containing cationic lipids, the
dried lipid film is resuspended in an appropriate solution such as sterile
water or
an isotonic buffer solution such as 10 mM Tris/NaCI, sonicated, and then the
preformed liposomes are mixed directly with the DNA. The liposome and DNA
form a very stable complex due to binding ofthe positively charged liposomes
to
the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are
prepared by a number of methods, well known in the art. Commonly used
methods include Ca2~-EDTA chelation (Papahadjopoulos etal., Biochim. Biophys.
Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); ether injection
(Deamer,
D. and Bangham, A., Biochim. Biophys. Acta (1976) 443:629; Ostro et al.,
Biochem. Biophys. Res. Commun. ( 1977) 76:836; Fraley et al., Proc. Natl.
Acad.
Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,
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Proc. Natl. Acad. Sci. USA ( 1979) 76:145); and reverse-phase evaporation
(REV)
(Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. and
Papahadjopoulos,
D., Proc. Natl. Acad. Sci. USA (1978) 75:145; Schaefer-Bidder et al., Science
( I 982) 215:166), which are herein incorporated by reference.
Generally, the ratio ofDNA to liposomes will be from about 10:1 to about
1:10. Preferably, the ration will be from about 5: I to about 1: 5. More
preferably,
the ratio will be about 3:1 to about 1:3. Still more preferably, the ratio
will be
about 1:1.
U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
reports on the injection of genetic material, complexed with cationic
liposomes
carriers, into mice. U.S. PatentNos. 4,897,355, 4,946,787, 5,049,386,
5,459,127,
5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no.
WO 94/29469 (which are herein incorporated by reference) provide cationic
lipids
for use in transfecting DNA into cells and mammals. U. S. Patent Nos. 5,
589,466,
5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/29469
(which are herein incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a
retroviral particle containing RNA which comprises a sequence encoding MI5.
Retroviruses from which the retroviral plasmid vectors may be derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus,
Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape
leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines
to form producer cell lines. Examples of packaging cells which may be
transfected
include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as
described in Miller, Human Gene Therapy I:5-14 (1990), which is incorporated
herein by reference in its entirety. The vector may transduce the packaging
cells
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through any means known in the art. Such means include, but are not limited
to,
electroporation, the use of liposomes, and CaP04 precipitation. In one
alternative,
the retroviral plasmid vector may be encapsulated into a liposome, or coupled
to
a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which
include polynucleotide encoding MIS. Such retroviral vector particles then may
be employed, to transduce eukaryotic cells, either in vitro or in vivo. The
transduced eukaryotic cells will express MIS.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with
MIS polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated such that it encodes and expresses MIS, and at the same time is
inactivated in terms of its ability to replicate in a normal lytic viral life
cycle.
Adenovirus expression is achieved without integration of the viral DNA into
the
host cell chromosome, thereby alleviating concerns about insertional
mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines for many
years
with an excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.
Respir.
Dis.109:233-238). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin
and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. ( 1991 )
.Science
252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive
studies to attempt to establish adenovirus as a causative agent in human
cancer
were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad Sci. USA
76:6606).
Suitable adenoviral vectors useful in the present invention are described,
for example, in Kozarsky and Wilson, Curr. Opirz Genet. Devel. 3:499-503
(1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human
Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994);
Wilson et al., Nature 365:691-692 ( 1993); and U. S. Patent No. 5,652,224,
which
are herein incorporated by reference. For example, the adenovirus vector Ad2
is
useful and can be grown in human 293 cells. These cells contain the E1 region
of
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adenovirus and constitutively express Ela and Elb, which complement the
defective adenoviruses by providing the products of the genes deleted from the
vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and
Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus
and/or packaging cell line to form infectious particles. The resulting virus
is
capable of infecting cells and can express a polynucleotide of interest which
is
operably linked to a promoter, but cannot replicate in most cells. Replication
deficient adenoviruses may be deleted in one or more of all or a portion of
the
following genes: E 1 a, E 1 b, E3, E4, E2a, or L 1 through LS .
In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
N.,
Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors containing
as
little as 300 base pairs of AAV can be packaged and can integrate, but space
for
exogenous DNA is limited to about 4.5 kb. Methods for producing and using
such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941,
5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention
will include all the sequences necessary for DNA replication, encapsidation,
and
host-cell integration. The MIS polynucleotide construct is inserted into the
AAV
vector using standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press ( 1989). The
recombinant AAV vector is then transfected into packaging cells which are
infected with a helper virus, using any standard technique, including
lipofection,
electroporation, calcium phosphate precipitation, etc. Appropriate helper
viruses
include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
Once the packaging cells are transfected and infected, they will produce
infectious
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AAV viral particles which contain the MIS polynucleotide construct. These
viral
particles are then used to transduce eukaryotic cells, either ex vivo or in
vivo. The
transduced cells will contain the MIS polynucleotide construct integrated into
its
genome, and will express MIS.
Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide sequences (e.g.
encoding MIS) via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411,
published September 26, 1996; International Publication No. WO 94/12650,
published August 4, 1994; Koller etal., Proc. Natl. Acad. Sci. USA 86:8932-
8935
( 1989); and Zijlstra et al. , Nature 342:43 S-43 8 ( 1989). This method
involves the
activation of a gene which is present in the target cells, but which is not
normally
expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in
the art, which contain the promoter with targeting sequences flanking the
promoter. Suitable promoters are described herein. The targeting sequence is
sufficiently complementary to an endogenous sequence to permit homologous
recombination ofthe promoter-targeting sequence with the endogenous sequence.
The targeting sequence will be sufficiently near the 5' end of the MIS desired
endogenous polynucleotide sequence so the promoter will be operably linked to
the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the
S' and 3' ends. Preferably, the 3' end of the first targeting sequence
contains the
same restriction enzyme site as the 5' end of the amplified promoter and the
5' end
of the second targeting sequence contains the same restriction site as the 3'
end of
the amplified promoter. The amplified promoter and targeting sequences are
digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either
as naked polynucleotide, or in conjunction with transfection-facilitating
agents,
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such as liposomes, viral sequences, viral particles, whole viruses,
lipofection,
precipitating agents, etc., described in more detail above. The P promoter
targeting sequence can be delivered by any method, included direct needle
injection, intravenous injection, topical administration, catheter infusion,
particle
accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells.
Homologous recombination between the construct and the endogenous sequence
takes place, such that an endogenous MIS sequence is placed under the control
ofthe promoter. The promoter then drives the expression ofthe endogenous MIS
sequence.
Preferably, the polynucleotide encoding MIS contains a secretory signal
sequence that facilitates secretion of the protein. Typically, the signal
sequence
is positioned in the coding region of the polynucleotide to be expressed
towards
or at the 5' end of the coding region. The signal sequence may be homologous
or
heterologous to the polynucleotide of interest and may be homologous or
heterologous to the cells to be transfected. Additionally, the signal sequence
may
be chemically synthesized using methods known in the art.
Any mode of administration of any ofthe above-described polynucleotides
constructs can be used so long as the mode results in the expression of one or
more molecules in an amount sufficient to provide a therapeutic effect. This
includes direct needle injection, systemic injection, catheter infusion,
biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots,
other
commercially available depot materials, osmotic pumps (e.g., Alza minipumps),
oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and
decanting or topical applications during surgery. For example, direct
injection of
naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or
a
protein-coated plasmid into the portal vein has resulted in gene expression of
the
foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
A preferred method of local administration is by direct injection.
Preferably, a recombinant molecule of the present invention complexed with a
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delivery vehicle is administered by direct injection into or locally within
the area
of arteries. Administration of a composition locally within the area of
arteries
refers to injecting the composition centimeters and preferably, millimeters
within
arteries.
Another method of local administration is to contact a polynucleotide
construct of the present invention in or around a surgical wound. For example,
a
patient can undergo surgery and the polynucleotide construct can be coated on
the
surface of tissue inside the wound or the construct can be injected into areas
of
tissue inside the wound.
Therapeutic compositions useful in systemic administration include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
administration comprise liposomes comprising ligands for targeting the vehicle
to
a particular site.
Preferred methods of systemic administration, include intravenous
injection, aerosol, oral and percutaneous (topical) delivery. Intravenous
injections
can be performed using methods standard in the art. Aerosol delivery can also
be
performed using methods standard in the art (see, for example, Stribling et
al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281 (1992), which is incorporated
herein by reference). Oral delivery can be performed by complexing a
polynucleotide construct of the present invention to a carrier capable of
withstanding degradation by digestive enzymes in the gut of an animal.
Examples
of such carriers, include plastic capsules or tablets, such as those known in
the art.
Topical delivery can be performed by mixing a polynucleotide construct of the
present invention with a lipophilic reagent (e.g., DMSO) that is capable of
passing
into the skin.
Determining an ef~'ective amount of substance to be delivered can depend
upon a number of factors including, for example, the chemical structure and
biological activity of the substance, the age and weight of the animal, the
precise
condition requiring treatment and its severity, and the route of
administration.
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The frequency of treatments depends upon a number of factors, such as the
amount of polynucleotide constructs administered per dose, as well as the
health
and history of the subject. The precise amount, number of doses, and timing of
doses will be determined by the attending physician
Phenotypes of genetically altered adult mice have yielded several clues for
possible role for MI5 in the adult, including regulation of steroidogenesis.
Mice
chronically overexpressing a human MIS transgene develop varying degrees of
gonadal abnormalities in the adult (Behringer et al., Nature 345:167-170 (
1990)).
Soon after birth, ovaries become depleted of germ cells and organize into
structures resembling seminiferous tubules; later, the ovaries degenerate in
the
adult. Male mice (25%) from the highest MI5-overexpressing animals have
undescended testes, which are also depleted of germ cells. These males lacked
seminal vesicles and had underdeveloped epididymides, feminized external
genitalia, and serum levels of testosterone 1/lOth those in normal male mice
(Behringer et al., Nature 345:167-170 (1990); Lyet et al., Biol. Reprod.
52:444-
454 (1995)). These results suggested that MI5 overexpression might interfere
with androgen biosynthesis in Leydig cells. Conversely, homologous
recombination in mice so that they no longer expressed either the MI5 ligand
(Behringer et al., Cell 79:415-425 (1994)) or the MIS type II receptor
(Mishina
et al., Genes Dev. 10:2577-2587 (1996)) also resulted in gonadal abnormalities
consisting of Leydig cell hyperplasia and focal atrophy of the germinal
epithelium.
Thus, MIS appears to have a role in maintaining steroid hormone balance in
both
male and femal gonads after birth.
Leydig cells or intestinal cells are found in the testes surrounding the
seminiferous tubules. Their major function is to produce testosterone, which
is
essential for the normal male phenotype. Testosterone is synthesized from
cholesterol in five steps by the activity of four enzymes (FIG. 1 ), three of
which
the present inventors have studied: P450scc, P450c17, and 3~3-hydroxysteroid
dehydrogenate/OS-04-isomerase (3 (3HSD)(Payne and Youngblood, Biol. Reprod.
52:217-225 ( 1995)). P450cc (cytochrome P450-side chain cleavage, also known
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as CYP1 1A) is a member of the superfamily of cytochrome P450 hemeproteins
(Nelson et al., DNA Cell Biol. 12:1-51 (1993)), is located on the inner
mitochondrial membrane, and catalyzes the committed steps of cholesterol
conversion to steroid hormones by converting the 27-carbon cholesterol
molecule
to the 21-carbon pregnenolone. Pregnenolone moves out of the mitochondria and
is converted to progesterone by the activity of 3~3HSD, a nonP450 enzyme.
Cytochrome P450c17a hydroxylase/C"-zo lysase (P450C17, CYP17) has dual
activities; it hydroxylates progesterone at the 17a position and converts the
21-carbon 17a-hydroxyprogesterone to the 19 carbon androstenedione.
Androstenedione is then converted to testosterone by the activity of
17-ketosteroid reductase, a non-P450 enzyme the reduces the ketone at the
carbon 17 position.
Recent studies have shown that the steady state levels of messenger RMAs
(mRNAs) for steroidogenic enzymes P450scc, 3 ~3HSD and P450C 17 appear
down-regulated in the testes and in purified Leydig cells of the
MIS-overexpressing transgenic mice, as was the level of serum testosterone and
estradiol (Racine et al., Proc. NatlAcad. Sci. USA 95: 594-599 (1998);
Rouiller-
Fabre et al., Endocrinology 139:1213-1220 ( 1998)). Correlative PT-PCR results
showed that the MIS type II receptor mRNA was present in purified Leydig
cells,
suggesting that the MIS exterted its observed Leydig cell effects directly via
the
MIS receptor (Racine et al., Proc. Natl Acad. Sci. USA 95: 594-599 (1998)).
Signal transduction by members of the TGF~3 family of glycoprotein
homodimers occurs when the ligand binds to a heteromeric complex of single
transmembrance, serine/threonine kinases. Ligand specificity within the family
is
determined by the type II receptor, which, in turn, recruits and
phosphorylates the
appropriate type I receptor for subsequent downstream signaling via subsets of
ligand-specific Smads (Kretzschmar and Massague, Curr. Opin. Genet. Dev.
8:103-111 ( 1998)). Efforts to determine the molecular mechanisms of MIS
signal
transduction have led us and others to the cloning of the MIS ligand and its
MIS
type II receptor and their characterization (Cate et al., Cell 45:685-698
(1986);
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Picard et al., Proc. Natl. Acad. Sci. USA 83:5464-5468 (1986); Baarends et
al.,
Development 120:189-197 (1994); di Clemente, et al., Mol. Endocrinol. 8:1006-
1020 (1994); Teixeiraetal., Endocrinology 137:160-165 (1996)). To understand
the downstream pathways that are activated by the MIS ligand binding to its
receptor, we are dissecting the role that MIS plays in Leydig cell function
and
steroidogenesis. Using the rodent Leydig tumor cells lines R2C and MA-10, we
have established a system for studying MIS signal transduction and have been
able
to show that MIS regulates steroidogenesis at the transcriptional level.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
In the following examples, the materials and methods used are as follows.
Materials
Chemicals were obtained from Fisher Scientific (Fairlawn, NJ) or Sigma
Chemical Co. (St. Louis, MO) unless otherwise noted. Maymouth's MB752/1
medium, gentamicin, oligonucleotides, and trypsin-EDTAwere obtained from Life
Technologies, Inc. (Grand Island, NY). Restriction and modifying enzymes,
firefly luciferase and Renilla luciferase vectors, and reagents were purchased
from
Promega Corp. (Madison, WI). Radionucleotides were purchased from New
England Nuclear (Boston, MA). Rat P450scc complementary DNA (cDNA) was
a gift from Dr. J.S. Richards (Baylor College of Medicine, Houston, TX).
Female(fetal calf serum (FCS) was obtained from Aires Scientific/Biologos
(Richardson, TX). Standard recombinant DNA techniques were used (Sambrook
et al., Molecular Cloning. A LaboratoryManual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY ( 1989)). Advantage polymerase enzyme was used
in the PCR amplification (CLONTECH Laboratories, Inc., Palo Alto, CA). All
animal studies were performed in accordance with the NIH Guide for the Care
and
Use of Laboratory Animals.
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Cell Culture
R2C, a rat Leydig tumor cell line (American Type Culture Collection,
Manassas, VA), was maintained in F-10 medium containing 15% horse serum and
2.5% female FCS (Aires Scientific/Biologos). The mouse MA-10 Leydig rumor
cell line, a gift from Dr. Mario Ascoli (University of Iowa, Ames, IA), was
maintained in Waymouth's MB 752/1 medium (Life Technologies, Inc.) modified
to contain 20 mm HEPES (pH 7.4), 50 pg/ml gentamicin, and 15% horse serum.
For these experiments, R2C and MA-10 cells were incubated in a humidified
atmosphere with S% COZ at 37°C and fed every other day until reaching
80-90%
confluence.
Northern Analyses
Total RNA was extracted using the guanidine isothiocyanate-cesium
chloride method (Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). All
RNA was extracted with successive rounds of phenol, chloroform, and ether;
ethanol precipitated; and quantified by absorbance at Az6o. Ten micrograms of
each RNA sample were denatured with dimethylsulfoxide and glyoxal at 65
° C,
separated in a 1.5% agarose gel, blotted overnight onto nylon membranes, and
either baked at 80 ° C in a vacuum oven or UV cross-linked. Testes were
surgically extracted from 30-day postnatal rats and homogenized, and RNA was
extracted as before for use as a positive control. RNA from isolated primary
Leydig cells of21-day-old rats purified by Percoll density gradient
centrifugation
to 95% homogeneity was provided by Dr. Mary Lee (Lee et al., "Mullerian
inhibiting substance type II receptor expression and binding in primary Leydig
cells," Endocrinology (in press)). Blots were prehybridized with 100gg/ml
sonicated salmon sperm DNA in 50% formamide hybridization solution for 3 h at
65 ° C for riboprobes and at 42 ° C for random primed probes.
The MI S type II
receptor plasmid was linearlized with EcoRV and riboprobes made with SP6
polymerase using standard techniques. 32P-Labeled random primed cDNA probes
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were made for P450cc and 3 (3HSD. Rat P450scc cDNA was a gift from Dr. J. S.
Richards (Baylor College of Medicine). The P450c 17 plasmid was linearized
with
EcoRI and the 3zP-labeled riboprobe made with T7 RNA polymerase using
standard techniques. Blots were hybridized overnight with 2x106 cpm/ml probe
with riboprobes at 65 ° C and washed at 72 ° C with random
primed human probes
at 42°C and washed at 60°C with O. lx SSC (lx SSC=150 mM sodium
chloride
and 15 mM sodium citrate)-0.1% SDS and exposed to radiographic film with
intensifying screens at -70 ° C for 3 days. All animal studies were
performed in
accordance with the NIH Guide for the Care and Use of Laboratory Animals, with
institutional approval on 8-19-98 under accession no. 97-4216.
Radioimmunoassays
MA-10 cells were plated on day 0 in sextuplicate at Sx104/well in 12-well
plates for each time point indicated. Recombinant human MIS (rhMIS) was
prepared from Chinese hamster ovary (CHO) cells stable transected with a
linear
construct ofthe human MI5 gene (Cate et al., Cell 45:685-698 (1986)). Active
secreted protein in the growth medium was then passed over an immunoaffinity
column prepared with the monoclonal MIS antibody, 6E11, conjugated to
Affi-Gel-10 (Ragin, et al., Protein Expr. Purif. 3:236-245 (1992)). Bound MI5
was then eluted offthe column with 3M ammonium thiocyanate solution, pH 7.4,
or 1M acetic acid, pH 3Ø Protein concentrations were determined by Bradford
assays after pH neutralization and desalting by centrifugal filtration
(Bradford,
M.M., Anal Biochem. 72:248-254 (1976)). The bioactivity of the MI5 was then
verified using an established organ culture assay, which grades the regression
of
the 14.5-day gestation rate urogential ridge (Donohoe et al., J. Surg. Res.
23:141-
148 (1977)). Vehicle control or 105 nM MIS in the presence or absence of
(Bu)zcAMP (50 gm) was added to MA-10 cells 2 days after plating and incubated
for the indicated number of days. Culture medium was collected and assayed by
RIA for total accumulated progesterone and testosterone by the NICHHD P30
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Reproductive and Endocrine Sciences RIA Core Laboratory at the Massachusetts
General Hospital.
Luciferase Reporter Assays
The P450c17 promoter fragment of 1018 by was amplified by PCR from
mouse genomic DNA using 5'-GAGCTCGAGTATTGGCATTGCGTCCC and
5'-CTCGAGGGCAGATGGCCAGCTGTGGA as primers with complimentary
SacI and XhoI sites (Payne et al., J. Steroid Biochem. Mol. Biol 43:895-906
( 1992)). Advantage polymerase enzyme was used in the PCR amplification
(CLONTECH Laboratories, Inc., Palo Alto, CA). The PCR fragment was cloned
into pCRII (Invitrogen, San Diego, CA) for sequence analysis and cut out with
SacI/Xhol. PGL3B vector was digested with SacI and XhoI and ligated to the
P450c17 promoter fragment, generating P450c-17-Luc. The constructs were
purified by cesium chloride ultracentrifugation. MA-10 cells were plated at
2x105
in triplicate and were transected with P450c-17-a-Luc using the FuGene 6 lipid
protocol (Roche Molecular Biochemicals). PGL3B, the promoterless patent
plasmid was used as a negative control. pRL-TK encoding Renilla luciferase
(Promega Corp.) under the control of a HSV thymidine kinase promoter was
cotransfected as an internal control for transfection efficiency and steroid
pathway
specificity. Cell lysates were made and assayed for luciferase activity using
a
Berthold Automatic luminometer (Mallac, Inc., Turku, Finland).
Example 1
The MIS Type II Receptor Is Expressed in Leydig Cells
The MIS ligand binds to the MIS type II receptor to initiate downstream
signaling. Northern analysis (see FIG. 2) was performed to determine whether
there was requisite expression of the MIS type II receptor in total RNA
isolated
from rat testes, R2C cells, MA-10 cells, and purified primary Leydig cells.
These
findings indicate that Leydig cells could respond directly via the MIS type II
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receptor from paracrine production of MIS in the nearby Sertoli cells. The
bands
observed under stringent hybridization conditions in the cell lines and
purified
Leydig cells migrated a distance equal to that seen with testis RNA,
suggesting
that the MIS type II receptor probe hybridized to the same mRNA species.
MA-10 cells show a faster migrating band than that seen with R2C cells in
other
Northern experiments.
Example 2
MIS Inhibits Steroid Production by Leydig Cells
To determine whether MA-10 would serve as suitable cells in which to
study the down-regulation of steroid hormone production observed in mice
overexpressing MIS in vivo, MA-10 were incubated with MIS and the levels of
progesterone and testosterone secreted into the medium was measured (FIGS. 3A
and 3B). MA-10 cells are a mouse Leydig cell tumor line that, in addition,
have
functional gonadotropin receptors resulting in enhanced steroid production in
response to cAMP or LH/hCG, thus mimicking the physiological state of Leydig
cells in vivo (Payne and Youngblood, Biol. Reprod. 52:217-225 (1995); Ascoli,
M., Endocrinology 108:88-95 (1981)).
FIG. 3A shows that incubation of MA-10 cells with MIS for 2 days
resulted in a modest, but significant, 40% reduction in progesterone secretion
in
both the cAMP-stimulated and unstimulated states. FIG. 3B shows the
concentration oftestosterone secreted by MA-10 cells incubated with MIS over
a 2-day time course was 10-fold lower than that of cells not treated with MIS.
The level and time course of steroid hormone reduction were similar to those
seen
when human follicular cells, harvested at the time of in vivo fertilization,
were
incubated with recombinant MIS (Kim et al., J. Clin. Endocrinol. Metab. 75:911-
917 (1992)) and when primary Leydig cells were incubated with MIS (Rouiller-
Fabre et al., Endocrinology 139:1213-1220 (1998)).
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Example 3
MIS Reduces Steady State Levels of c17 mRNA
To uncover the molecular mechanisms responsible for these effects, the
effect of MIS on the steady state levels of the mRNAs for the steroidogenic
enzymes P450scc, 3~3HSD, and P450c17 were examined (FIGS. 4A-C).
Northern analysis revealed that the levels of mRNA, although marginally lower
for
P450scc and 3~3HSD for both R2C after 3-h treatment and MA-10 cells after
overnight treatment with MIS (FIGS. 4A and 4B, respectively), were
dramatically
reduced for P450c17 to undetectable levels in cAMP-stimulated MA-10 cells.
We also tested whether the down-regulation of c17 mRNA by MIS
required protein synthesis, which would indicate whether the effect of MIS on
c 17
mRNA was direct and found that overnight incubation of MA-10 cells with MIS
in the presence of cyclohexamide, a protein synthesis inhibitor, continued to
cause
a significant decrease in the steady state level of c17 mRNA (FIG. 4C). RNA
was
prepared from MA-10 cells, incubated with MIS in the presence or absence of
cyclohexamide and analyzed by Northern blot for c17 mRNA. Surprisingly,
addition of cyclohexamide alone was su~cient to induce expression of c 17 mRNA
above levels seen with cAMP induction.
Example 4
MIS Regulates c1 7 Expression at the Transcriptional Level
Changes in mRNA for the steroidogenic enzymes could be reflected in,
among others, differences in message stability, nuclear export, or
transcription, all
ofwhich could be potential points for regulation by MIS. To study
transcriptional
regulation of the Cypl7gene by MIS, DNA constructs were made containing
approximately lkb of the Cypl7 promoter fused with the firefly luciserase
gene,
transfected them into MA-10 cells, and assayed for luciferase activity (FIGS.
SA-
C). In the first set of experiments, on the day after transfection with the
reporter
construct, MA-10 cells were incubated with varying concentrations of MIS and
harvested either 18 or 29 h later to permit induction of luciferase activity,
which
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was measured and compared (FIG. 5A). MIS appears to exert its effect on the
exogenous Cypl7 promoter/luciferase reporter after only 18 h with 70 nM MIS.
By 29 h, 3 S nM MIS was able to markedly lower luciferase activity and, with
105
nM MIS, luciferase activity was reduced to background levels. These
concentrations, which are high compared with those required for
transcriptional
activation by other members of the TGF~i family, are similar to those required
for
an MIS effect in other assays and with the 35-nM MIS concentration required
for
complete regression of the Mullerian duct in the organ culture bioassay used
to
test the potency of MIS preparations (Racine et al., Proc. Natl Acad Sci. USA
95:594-599 (1998)s; MacLaughlin et al., Methods Enzymol. 198:358-369
( 1991 )). Also, firefly luciferase activation is normalized to a
cotransfected Renilla
reporter to rule out nonspecific effects on the cell by MIS. It is also
important to
note that the MIS preparations were tested and were found to be free of
activin
and TGF~3 by enzyme-linked immunoabsorbant assay, and that when TGF(3 was
added to MA-10, the Cypl7-driven luciferase activity was not significantly
different from that of untreated cells (not shown).
FIG. 5B shows that MIS incubation on the day after transfection has a
slight, but significant, effect on luciferase after 3 h and becomes pronounced
after
18 h. In another experiment, MA-10 cells when incubated with L9, an inactive
form of the MIS ligand that has been mutated so that pro-MIS could not be
cleaved to generate the bioactive C-terminal portion of the hormone
(MacLaughlin et al., Endocrinology 131:291-296 ( 1992)), did not affect
luciferase
activity as did bioactive, cleavable MIS at the same concentration and time
(shown
with an asterisk in FIG. 5B). As L9 is produced in CHO cells and purified in
the
same manner as wild-type MIS, we can conclude that the effect on P450c17
transcription is due to MIS and not to a possible copurified contaminant.
Luciferase expression appears maximal after overnight transfection, as
luciferase activity was not significantly different in the control MA-10 cells
with
3- to 18-h additional incubation (FIG. 5B). To take advantage of an earlier
MIS
effect, we transfected the Cypl7 reporter construct and added MIS at the same
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time. As shown in FIG. SC, 18h after transfection, we observed a 4-fold
decrease
in luciferase activity with added MIS, which is considerably greater than that
observed when MIS was added 1 day later. This observation suggests that MIS
can more effectively repress expression of the Cypl7 promoter-driver reporter
before it is fully activated.
In human males, after the first year of life there is a reciprocal
relationship
between expression of MIS and serum testosterone concentrations that remains
throughout life (Bardin et al., "Androgens," in Reproductive Endocrinology,
Surgery and Technology, Adashi et al., eds., Lippincott-Raven, Philadelphia
(1995), pp. 505-525; Josso et al., Early Hum. Dev. 33:91-99 (1993); Lee et
al.,
J. Clin. Endocrinol. Metab. 81:571-576 (1996)). During fetal life and again
after
the testosterone nadir in the immediate perinatal period, however, both MIS
and
testosterone levels are high. At birth, the levels of MIS in males are
slightly lower,
but still 10-fold higher than those in females. During the first 6 months of
life
(minipuberty), there is a sharp peak in serum testosterone and a gradual
increase
in MIS concentration to its highest level, which is reached by the first 12
months
of age when the concentration os serum testosterone in males is again at a low
point. In precocious puberty syndromes, such as occurs with testotoxicosis or
in
the McCune-Albright syndrome, testosterone levels are elevated at an early
age.
Testotoxicosis is caused by activating mutations in the stipulatory G
protein-coupled receptor (that are usually due to somatic mutations during
early
embryonic development) are responsible for the McCune-Albright syndrome (Yen
et al., Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical
Management, Saunders, Philadelphia (1999), pp. viii 839). As serum MIS and
testosterone concentrations are inversely related during normal and precocious
puberty, others have speculated that androgens regulate MIS expression (Rey et
al., J. Clin. Endocrinol. Metab. 77:1220-1226 (1993)). However, attempts to
simulate androgen-regulated MIS expression in tissue culture (Happ and P.K.
Donahoe, unpublished) have not been fruitful, and thus the hypothesis remains
speculative.
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We have observed an effect of MIS on progesterone secretion by MA-10
cells that was reflected in a decrease in steady state mRNA levels for both
P450scc and 3 (3HSD.
MIS regulation of steroidogenesis in Leydig cells by transcrptional control
of Cypl7 exposes a conundrum in MIS-mediated suppression of testosterone
synthesis that may indicate is developmentally regulated. Fetal Leydig cells
proliferate and show increased androgen synthesis, which is required for male
phenotypic development, independent of gonadotropin stimulation in the rodent
(O'Shaughnessy et al., Endocrinology 139:1141-1146 (1998); Kendall et al.,
Genes Dev. 9:2007-2019 (1995)). We would have expected that these fetal
Leydig cells must be refractory to MIS-mediated inhibition of androgen
synthesis,
because the level of MIS is also high at this time. However, in
MIS-overexpressing mice (Behringer et al., Nature 345:167-170 (1990))
impairment of the testosterone-regulated differentiation of the fetal Wolfian
duct,
which is the precursor ofthe vas deferens, epididymides, and seminal vesicles,
was
observed. Also, incubation of fetal rat Leydig cells with MIS in vitro results
in
suppression of testosterone synthesis (Rouiller-Fabre et al., Endocrinology
139:1213-1220 (1998)). After birth, when MIS levels remain high, fetal-derived
Leydig cells begin regressing, and lower levels of androgens ensue. The adult
Leydig cells arise and proliferate from mesenchyme precursors at puberty, when
MIS levels reach their nadir (Lee et al., J. Clin. Endocrinol. Metab. 81:571-
576
( 1996); Hudson et al., J. Clin. Endocrinol. Metab. 70:16-22 ( 1990)), and
begin
producing androgens after stimulation with LH (Saez, J.M., Endocr. Rev. I S:
574-
626 (1994)). The MIS-regulated cell lines used in our studies were originally
derived from this adult Leydig cell population. This distinction is important
when
attempting to understand why fetal Leydig cells, which are in an environment
with
high levels of MIS, continue to produce androgens.
Although the amount of testosterone secreted by MA-10 cells is much
lower than that secreted by purified Leydig cells (Rouiller-Fabre et al.,
Endocrinology 139:1213-1220 (1998)) and significantly lower than the level of
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progesterone, prevailing opinion is that MA-10 cells do not produce
testosterone.
Our results clearly indicate not only that MA-10 cells secrete measurable
amounts
of testosterone into the medium, but that it can be enhanced 3-fold by cAMP
addition. The original report detailing the cloning and characterization of
the
MA-10 cell line also shows low levels of testosterone secretion, which was
induced with hCG. MA-10 cells have proven enormously valuable to understand
MIS-mediated inhibition of Cyp 17 expression and will be very useful in our
future
efforts to understand the molecular mechanisms of that inhibition.
In our studies to determine whether the MIS signal transduction directly
mediated suppression of Cyp 17 mRNA expression in the absence of protein
synthesis, we observed that Cyclohexamide alone was able to induce expression
of Cyp 17 mRNA. The increase in Cyp 17 mRNA with cyclohexamide treatment
is probably not artifactual and is commonly observed with labile mRNAs
(Theodorakis and Cleveland, in Translation Control, Hershey et al., eds., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1996), pp. 631-652);
it could indicate the presence of a protein that inactivates or turns over a
crucial
component of Cypl7 expression. In the case of c-myc mRNA, mRNA stability
has been linked to translation of the c-myc mRNA itself, i. e., c-myc mRNA
decay
is accelerated by its translation. Cyclohexamide blocks c-myc translation and
therefore prolongs the c-myc mRNA half life (Yeilding et al., J. Biol. Chem.
273:15749-15757 (1998)).
Many advances in understanding TGF~3 signal transduction mechanisms
have been made because of the availability of both a cell line that responds
to
TGF~3 and a reporter gene with which to measure that response. Until now such
a model system for studying MIS signal transduction has been lacking. Evidence
of transcriptional regulation of steroidogenic enzymes in rodent Leydig cells
by
MIS is a significant advance in our understanding of MIS signal transduction.
This first example of transcriptional regulation of a gene by MIS can be used
to
advantage to dissect and define the relevant MIS-specific cis-elements and
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traps-acting factors and the upstream pathways that are responsible for MIS
receptor-mediated molecular events.
There are a number of potential clinical implications that could emanate
from the study of this MIS signal transduction pathway. For example,
MIS-specific checkpoints in the MIS-mediated down-regulation of testosterone
synthesis could be activated and used to lower endogenous testosterone in such
clinical settings as prostatic cancer and benign prostatic hypertrophy or to
lower
elevated steroids in precocious puberty syndromes. Current treatments with
GnRH long acting analogs to down-regulate the GnRH receptor have been
successful in the treatment of central precocious puberty (Hoffman and
Crowley,
N. Engl. J. Med. 307:1237-1241 (1982)), but it is the downstream variants for
which treatment is not yet optimal and could be augmented by MIS-related
treatments. Over 60% of McCune-Albright patients, who for unknown reasons
have a 31 ratio of females to males, have elevated sex steroids in the absence
of
elevated gonadotropins due to activating mutations in the a-subunit of the G
protein-regulating adenylyl cyclase (Ringel etal., Medicine 75:171-184
(1996)).
In these patients with gonadotropin-independent precocious puberty,
suppression
of multiple enzymes in the steroid production pathway by MIS might, for
example,
be used to augment the currently used aromatase inhibitor, which have been
helpful over the short term, but less effective over the long term ( 1-3 yr. )
(Feuillan
et al., N. Engl. J. Med. 315:1115-1119 (1986); Hauffaetal., Helv. Paediatr.
Acta
42:471-480 (1987)).
Example S
MIS Regulates Testosterone In Vivo
Adult male rats (250 g) were administered either hCG (50 U, n=3, white
bar), or 50 U hCG and 1 mg MIS (n=3, black bar) intraperitoneally. After 18
hours, serum was collected from each of the animals and tested for
testosterone
concentration using a radioimmunoassay. The results are shown in FIG. 6. The
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mean values in each group are shown, and error bars represent SEM, p<Ø05.
These data show that serum testosterone is lowered by MIS administration.
Example 6
Acute Administration of MIS Regulates Testosterone In hivo
To study the acute regulation of steroids, LH or hCG, with or without
rhMIS, is administered to animals by intraperitoneal injection. At six time
points
(10 animals each), animals are injected intraperitoneally with LH (25 IU) with
or
without MIS in a single injection. At the inidcated times (30 min, 1h, 2h, 4h,
8h,
18h), blood is collected for measurement of serum MIS, LH and testosterone,
and
the testes are harvested for preparation of RNA and for histology. mRNA
expression of steroidogenic enzymes (SCC, 3(3HSD, Cypl7, etc.) is analyzed by
Northern analysis. rhMIS suppresses serum testosterone concentration.
Example 7
Long-Term Administration of MIS Regulates Testosterone
In Yivo
To study the long term regulation of steroids, LH or hCG, with or without
rhMIS, is administered to animals via Alzet osmotic minipumps. The effects of
chronic MIS exposure is analyzed daily for one week. LH levels are measured,
since low testosterone will elevate LH, which in turn could increase the
testosterone concentration, to compensate for and possibly obscure an MIS
effect.
Conversely, LH or cAMP and subsequently high testosterone levels may be
required to observe the MIS inhibitory effects, as with the cAMP stimulation
of
MA-10 cells in vitro. IfLH is found to compensate for the MIS effect in vivo,
the
studies are repeated, in either hypophysectomized rats or animals
physiologically
clamped with a long acting LHRH analog, with subsequent carefully controlled
hCG or LH delivery prior to MIS treatment. rhMIS suppresses serum
testosterone concentration.
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It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are
possible in light of the above teachings and, therefore, are within the scope
of the
appended claims.
The entire disclosure of all publications (including patents, patent
applications, journal articles, laboratory manuals, books, or other documents)
cited herein are hereby incorporated by reference.
