Methods of treating musculodegenerative disease with an antibody that
binds growth differentiation factor-8
United States Patent: 7,534,432
Issued: May 19, 2009
Inventors: Lee; Se-Jin
(Baltimore, MD), McPherron; Alexandra C. (Baltimore, MD)
Assignee: The Johns Hopkins
University School of Medicine (Baltimore, MD)
Appl. No.: 11/700,267
Filed: January 29, 2007
Pharm Bus Intell
& Healthcare Studies
Growth differentiation factor-8 (GDF-8)
is disclosed along with its polynucleotide sequence and amino acid
sequence. Also disclosed are diagnostic and therapeutic methods of using
the GDF-8 polypeptide and polynucleotide sequences.
Description of the
SUMMARY OF THE INVENTION
The present invention provides a cell growth and differentiation factor,
GDF-8, a polynucleotide sequence which encodes the factor, and antibodies
which are immunoreactive with the factor. This factor appears to relate to
various cell proliferative disorders, especially those involving those
involving muscle, nerve, and adipose tissue.
Thus, in one embodiment, the invention provides a method for detecting a
cell proliferative disorder of muscle, nerve, or fat origin and which is
associated with GDF-8. In another embodiment, the invention provides a
method for treating a cell proliferative disorder by suppressing or
enhancing GDF-8 activity.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a growth and differentiation factor. GDF-8
and a polynucleotide sequence encoding GDF-8. GDF-8 is expressed at highest
levels in muscle and at lower levels in adipose tissue. In one embodiment,
the invention provides a method for detection of a cell proliferative
disorder of muscle, nerve, or fat origin which is associated with GDF-8
expression. In another embodiment, the invention provides a method for
treating a cell proliferative disorder by using an agent which suppresses or
enhances GDF-8 activity.
The TGF-.beta. superfamily consists of multifunctional polypeptides that
control proliferation, differentiation, and other functions in many cell
types. Many of the peptides have regulatory, both positive and negative,
effects on other peptide growth factors. The structural homology between the
GDF-8 protein of this invention and the members of the TGF-.beta. family,
indicates that GDF-8 is a new member of the family of growth and
differentiation factors. Based on the known activities of many of the other
members, it can be expected that GDF-8 will also possess biological
activities that will make it useful as a diagnostic and therapeutic reagent.
In particular, certain members of this superfamily have expression patterns
or possess activities that relate to the function of the nervous system. For
example, the inhibins and activins have been shown to be expressed in the
brain (Meunier, et al., Proc. Natl. Acad. Sci., USA, 85:247, 1988; Sawchenko,
et al., Nature, 334:615, 1988), and activin has been shown to be capable of
functioning as a nerve cell survival molecule (Schubert, et al., Nature,
344:868, 1990). Another family member, namely, GDF-1, is nervous
system-specific in its expression pattern (Lee, S. J., Proc. Natl. Acad. Sci.,
USA, 88:4250, 1991), and certain other family members, such as Vgr-1 (Lyons,
et al., Proc. Natl. Acad. Sci., USA, 86:4554, 1989; Jones, et al.,
Development, 111:531, 1991) OP-1 (Ozkaynak, et al., J. Biol. Chem.,
267:25220, 1992), and BMP-4 (Jones, et al., Development, 111:531, 1991). are
also known to be expressed in the nervous system. Because it is known that
skeletal muscle produces a factor or factors that promote the survival of
motor neurons (Brown, Trends Neurosci., 7:10, 1984), the expression of GDF-8
in muscle suggests that one activity of GDF-8 may be as a trophic factor for
neurons. In this regard, GDF-8 may have applications in the treatment of
neurodegenerative diseases, such as amyotrophic lateral sclerosis, or in
maintaining cells or tissues in culture prior to transplantation.
GDF-8 may also have applications in treating disease processes involving
muscle, such as in musculodegenerative diseases or in tissue repair due to
trauma. In this regard, many other members of the TGF-.beta. family are also
important mediators of tissue repair. TGF-.beta. has been shown to have
marked effects on the formation of collagen and to cause a striking
angiogenic response in the newborn mouse (Roberts, et al., Proc. Natl. Acad.
Sci., USA 83:4167, 1986). TGF-.beta. has also been shown to inhibit the
differentiation of myoblasts in culture (Massague, et al., Proc. Natl. Acad.
Sci., USA 83:8206, 1986). Moreover, because myoblast cells may be used as a
vehicle for delivering genes to muscle for gene therapy, the properties of
GDF-8 could be exploited for maintaining cells prior to transplantation or
for enhancing the efficiency of the fusion process.
The expression of GDF-8 in adipose tissue also raises the possibility of
applications for GDF-8 in the treatment of obesity or of disorders related
to abnormal proliferation of adipocytes. In this regard, TGF-.beta. has been
shown to be a potent inhibitor of adipocyte differentiation in vitro (Ignotz
and Massague, Proc. Natl. Acad. Sci., USA 82:8530, 1985).
The term "substantially pure" as used herein refers to GDF-8 which is
substantially free of other proteins, lipids, carbohydrates or other
materials with which it is naturally associated. One skilled in the art can
purify GDF-8 using standard techniques for protein purification. The
substantially pure polypeptide will yield a single major band en a
non-reducing polyacrylamide gel. The purity of the GDF-8 polypeptide can
also be determined by amino-terminal amino acid sequence analysis. GDF-8
polypeptide includes functional fragments of the polypeptide, as long as the
activity of GDF-8 remains. Smaller peptides containing the biological
activity of GDF-8 are included in the invention.
The invention provides polynucleotides encoding the GDF-8 protein. These
polynucleotides include DNA, cDNA and RNA sequences which encode GDF-8. It
is understood that all polynucleotides encoding all or a portion of GDF-8
are also included herein, as long as they encode a polypeptide with GDF-8
activity. Such polynucleotides include naturally occurring, synthetic, and
intentionally manipulated polynucleotides. For example, GDF-8 polynucleotide
may be subjected to site-directed mutagenesis. The polynucleotide sequence
for GDF-8 also includes antisense sequences. The polynucleotides of the
invention include sequences that are degenerate as a result of the genetic
code. There are 20 natural amino acids, most of which are specified by more
than one codon. Therefore, all degenerate nucleotide sequences are included
in the invention as long as the amino acid sequence of GDF-8 polypeptide
encoded by the nucleotide sequence is functionally unchanged.
Specifically disclosed herein is a genomic DNA sequence containing a portion
of the GDF-8 gene. The sequence contains an open reading frame corresponding
to the predicted C-terminal region of the GDF-8 precursor protein. The
encoded polypeptide is predicted to contain two potential proteolytic
processing sites (KR and RR). Cleavage of the precursor at the downstream
site would generate a mature biologically active C-terminal fragment of 109
amino acids with a predicted molecular weight of approximately 12,400. Also,
disclosed are full length murine and human GDF-8 cDNA sequences. The murine
pre-pro-GDF-8 protein is 376 amino acids in length, which is encoded by a
2676 base pair nucleotide sequence, becinning at nucleotide 104 and
extending to a TGA stop codon at nucleotide 1232. The human GDF-8 protein is
375 amino acids and is encoded by a 2743 base pair sequence, with the open
reading frame beginning at nucleotide 59 and extending to nucleotide 1184.
The C-terminal region of GDF-8 following the putative proteolytic processing
site shows significant homology to the known members of the TGF-.beta.
superfamily. The GDF-8 sequence contains most of the residues that are
highly conserved in other family members (see FIG. 3, see Original Patent).
Like the TGF-.beta. and inhibin .beta.s, GDF-8 contains an extra pair of
cysteine residues in addition to the 7 cysteines found in virtually all
other family members. Among the known family members, GDF-8 is most
homologous to Vgr-1 (45% sequence identity) (see FIG. 4, see Original Patent).
Minor modifications of the recombinant GDF-8 primary amino acid sequence may
result in proteins which have substantially equivalent activity as compared
to the GDF-8 polypeptide described herein. Such modifications may be
deliberate, as by site-directed mutagenesis, or may be spontaneous. All of
the polypeptides produced by these modifications are included herein as long
as the biological activity of GDF-8 still exists. Further, deletion of one
or more amino acids can also result in a modification of the structure of
the resultant molecule without significantly altering its biological
activity. This can lead to the development of a smaller active molecule
which would have broader utility. For example, one can remove amino or
carboxy terminal amino acids which are not required for GDF-8 biological
The nucleotide sequence encoding the GDF-8 polypeptide of the invention
includes the disclosed sequence and conservative variations thereof. The
term "conservative variation" as used herein denotes the replacement of an
amino acid residue by another, biologically similar residue. Examples of
conservative variations include the substitution of one hydrophobic residue
such as isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, such as the substitution of
arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine,
and the like. The term "conservative variation" also includes the use of a
substituted amino acid in place of an unsubstituted parent amino acid
provided that antibodies raised to the substituted polypeptide also
immunoreact with the unsubstituted polypeptide.
DNA sequences of the invention can be obtained by several methods. For
example, the DNA can be isolated using hybridization techniques which are
well known in the art. These include, but are not limited to: 1)
hybridization of genomic or cDNA libraries with probes to detect homologous
nucleotide sequences, 2) polymerase chain reaction (PCR) on genomic DNA or
cDNA using primers capable of annealing to the DNA sequence of interest, and
3) antibody screening of expression libraries to detect cloned DNA fragments
with shared structural features.
Preferably the GDF-8 polynucleotide of the invention is derived from a
mammalian organism, and most preferably from a mouse, rat, or human.
Screening procedures which rely on nucleic acid hybridization make it
possible to isolate any gene sequence from any organism, provided the
appropriate probe is available. Oligonucleotide probes, which correspond to
a part of the sequence encoding the protein in question, can be synthesized
chemically. This requires that short, oligopeptide stretches of amino acid
sequence must be known. The DNA sequence encoding the protein can be deduced
from the genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction when the
sequence is degenerate. This includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is preferably
performed on either single-stranded DNA or denatured double-stranded DNA.
Hybridization is particularly useful in the detection of cDNA clones derived
from sources where an extremely low amount of mRNA sequences relating to the
polypeptide of interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it is
possible, for example, to allow the autoradiographic visualization of a
specific cDNA clone by the hybridization of the target DNA to that single
probe in the mixture which is its complete complement (Wallace, et al., Nucl.
Acid Res., 9:879, 1981).
The development of specific DNA sequences encoding GDF-8 can also be
obtained by: 1) isolation of double-stranded DNA sequences from the genomic
DNA; 2) chemical manufacture of a DNA sequence to provide the necessary
codons for the polypeptide of interest; and 3) in vitro synthesis of a
double-stranded DNA sequence by reverse transcription of mRNA isolated from
a eukaryotic donor cell. In the latter case, a double-stranded DNA
complement of mRNA is eventually formed which is generally referred to as
Of the three above-noted methods for developing specific DNA sequences for
use in recombinant procedures, the isolation of genomic DNA isolates is the
least common. This is especially true when it is desirable to obtain the
microbial expression of mammalian polypeptides due to the presence of
The synthesis of DNA sequences Is frequently the method of choice when the
entire sequence of amino acid residues of the desired polypeptide product is
known. When the entire sequence of amino acid residues of the desired
polypeptide is not known, the direct synthesis of DNA sequences is net
possible and the method of choice is the synthesis of cDNA sequences. Among
the standard procedures for isolating cDNA sequences of interest is the
formation of plasmid- or phage-carrying cDNA libraries which are derived
from reverse transcription of mRNA which is abundant in donor cells that
have a high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare expression products can be
cloned. In those cases where significant portions of the amino acid sequence
of the polypeptide are known, the production of labeled single or
double-stranded DNA or RNA probe sequences duplicating a sequence putatively
present in the target cDNA may be employed in DNA/DNA hybridization
procedures which are carried out on cloned copies of the cDNA which have
been denatured into a single-stranded form (Jay, et al., Nucl. Acid Res.,
A cDNA expression library, such as lambda gt11. can be screened indirectly
for GDF-8 peptides having at least one epitope, using antibodies specific
for GDF-8. Such antibodies can be either polyclonally or monoclonally
derived and used to detect expression product indicative of the presence of
DNA sequences encoding GDF-8 can be expressed in vitro by DNA transfer into
a suitable host cell. "Host cells" are cells in which a vector can be
propagated and its DNA expressed. The term also includes any progeny of the
subject host cell. It is understood that all progeny may not be identical to
the parental cell since there may be mutations that occur during
replication. However, such progeny are included when the term "host cell" is
used. Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
In the present invention, the GDF-8 polynucleotide sequences may be inserted
into a recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle known in the art that
has been manipulated by insertion or incorporation of the GDF-8 genetic
sequences. Such expression vectors contain a promoter sequence which
facilitates the efficient transcription of the inserted genetic sequence of
the host. The expression vector typically contains an origin of replication,
a promoter, as well as specific genes which allow phenotypic selection of
the transformed cells. Vectors suitable for use in the present invention
include, but are not limited to the T7-based expression vector for
expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987). the pMSXND
expression vector for expression in mammalian cells (Lee and Nathans, J.
Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression
in insect cells. The DNA segment can be present in the vector operably
linked to regulatory elements, for example, a promoter (e.g., T7,
metallothionein I, or polyhedrin promoters).
Polynucleotide sequences encoding GDF-8 can be expressed in either
prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and
mammalian organisms. Methods of expressing DNA sequences having eukaryotic
or viral sequences in prokaryotes are well known in the art. Biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host are known in the art. Such vectors are used to
incorporate DNA sequences of the invention. Preferably, the mature
C-terminal region of GDF-8 is expressed from a cDNA clone containing the
entire coding sequence of GDF-8. Alternatively, the C-terminal portion of
GDF-8 can be expressed as a fusion protein with the pro-region of another
member of the TGF-.beta. family or co-expressed with another pro-region (see
for example, Hammonds, et al., Molec. Endocrin. 5:149, 1991; Gray, A., and
Mason, A., Science, 247:1328, 1990).
Transformation of a host cell with recombinant DNA may be carried out by
conventional techniques as are well known to those skilled in the art. Where
the host is prokaryotic, such as E. coli, competent cells which are capable
of DNA uptake can be prepared from cells harvested after exponential growth
phase and subsequently treated by the CaCl.sub.2 method using procedures
well known in the an. Alternatively. MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of the host
cell if desired.
When the host is a eukaryote, such methods of transfection of DNA as calcium
phosphate co-precipitates, conventional mechanical procedures such as
microinjection, electroporation, insertion of a plasmid encased in liposomes,
or virus vectors may be used. Eukaryotic cells can also be cotransformed
with DNA sequences encoding the GDF-8 of the invention, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes simplex
thymidine kinase gene. Another method is to use a eukaryotic viral vector,
such as simian virus 40 (SV40) or bovine papilloma virus, to transiently
infect or transform eukaryotic cells and express the protein, (see for
example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman
Isolation and purification of microbial expressed polypeptide, or fragments
thereof, provided by the invention, may be carried out by conventional means
including preparative chromatography and immunological separations involving
monoclonal or polyclonal antibodies.
The invention includes antibodies immunoreactive with GDF-8 polypeptide or
functional fragments thereof. Antibody which consists essentially of pooled
monoclonal antibodies with differer epitopic specificities, as well as
distinct monoclonal antibody preparations are provided. Monoclonal
antibodies are made from antigen containing fragments of the protein by
methods well known to those skilled in the art (Kohler, et al., Nature,
256:495, 1975). The term antibody as used in this invention is meant to
include intact molecules as well as fragments thereof, such as Fab and F(ab').sub.2,
which are capable of binding an epitopic determinant on GDF-8.
The term "cell-proliferative disorder" denotes malignant as well as
non-malignant cell populations which often appear to differ from the
surrounding tissue both morphologically and genotypically. Malignant cells
(i.e. cancer) develop as a result of a multistep process. The GDF-8
polynucleotide that is an antisense molecule is useful in treating
malignancies of the various organ systems, particularly, for example, cells
in muscle or adipose tissue. Essentially, any disorder which is
etiologically linked to altered expression of GDF-8 could be considered
susceptible to treatment with a GDF-8 suppressing reagent. One such disorder
is a malignant cell proliferative disorder, for example.
The invention provides a method for detecting a cell proliferative disorder
of muscle or adipose tissue which comprises contacting an anti-GDF-8
antibody with a cell suspected of having a GDF-8 associated disorder and
detecting binding to the antibody. The antibody reactive with GDF-8 is
labeled with a compound which allows detection of binding to GDF-8. For
purposes of the invention, an antibody specific for GDF-8 polypeptide may be
used to detect the level of GDF-8 in biological fluids and tissues. Any
specimen containing a detectable amount of antigen can be used. A preferred
sample of this invention is muscle tissue. The level of GDF-8 in the suspect
cell can be compared with the level in a normal cell to determine whether
the subject has a GDF-8-associated cell proliferative disorder. Preferably
the subject is human.
The antibodies of the invention can be used in any subject in which it is
desirable to administer in vitro or in vivo immunodiagnosis or
immunotherapy. The antibodies of the invention are suited for use, for
example, in immuno-assays in which they can be utilized in liquid phase or
bound to a solid phase carrier. In addition, the antibodies in these
immunoassays can be detectably labeled in various ways. Examples of types of
immunoassays which can utilize antibodies of the invention are competitive
and non-competitive immunoassays in either a direct or indirect format.
Examples of such immunoassays are the radioimmunoassay (RIA) and the
sandwich (immunometric) assay. Detection of the antigens using the
antibodies of the invention can be done utilizing immunoassays which are run
in either the forward, reverse, or simultaneous modes, including
immunohistochemical assays on physiological samples. Those of skill in the
art will know, or can readily discern, other immunoassay formats without
The antibodies of the invention can be bound to many different carriers and
used to detect the presence of an antigen comprising the polypeptide of the
invention. Examples of well-known carriers include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, agaroses and magnetite. The nature of the
carrier can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for binding
antibodies, or will be able to ascertain such, using routine
There are many different labels and methods of labeling known to those of
ordinary skill in the art. Examples of the types of labels which can be used
in the present invention include enzymes, radioisotopes, fluorescent
compounds, colloidal metals, chemiluminescent compounds, phosphorescent
compounds and bioluminescent compounds. Those of ordinary skill in the art
will know of other suitable labels for binding to the antibody, or will be
able to ascertain such, using routine experimentation.
Another technique which may also result in greater sensitivity consists of
coupling the antibodies to low molecular weight haptens. These haptens can
then be specifically detected by means of a second reaction. For example, it
is common to use such haptens as biotin, which reacts with avidin, or
dinitrophenyl, puridoxal, and fluorescein, which can react with specific
In using the monoclonal antibodies of the invention for the in vivo
detection of antigen, the detectably labeled antibody is given a dose which
is diagnostically effective. The term "diagnostically effective" means that
the amount of detectably labeled monoclonal antibody is administered in
sufficient quantity to enable detection of the site having the antigen
comprising a polypeptide of the invention for which the monoclonal
antibodies are specific.
The concentration of detectably labeled monoclonal antibody which is
administered should be sufficient such that the binding to those cells
having the polypeptide is detectable compared to the background. Further, it
is desirable that the detectably labeled monoclonal antibody be rapidly
cleared from the circulatory system in order to give the best
target-to-background signal ratio.
As a rule, the dosage of detectably labeled monoclonal antibody for in vivo
diagnosis will vary depending on such factors as age, sex, and extent of
disease of the individual. Such dosages may vary, for example, depending on
whether multiple injections are given, antigenic burden, and other factors
known to those of skill in the art.
For in vivo diagnostic imaging, the type of detection instrument available
is a major factor in selecting a given radioisotope. The radioisotope chosen
must have a type of decay which is detectable for a given type of
instrument. Still another important factor in selecting a radioisotope for
in vivo diagnosis is that deleterious radiation with respect to the host is
minimized. Ideally, a radio-isotope used for in vivo imaging will lack a
particle emission, but produce a large number of photons in the 140-250 keV
range, which may readily be detected by conventional gamma cameras.
For in vivo diagnosis radioisotopes may be bound to immunoglobulin either
directly or indirectly by using an intermediate functional group.
Intermediate functional groups which often are used to bind radioisotopes
which exist as metallic ions to immunoglobulins are the bifunctional
chelating agents such as diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules. Typical
examples of metallic ions which can be bound to the monoclonal antibodies of
the invention are .sup.111In, .sup.97Ru, .sup.67Ga, .sup.68Ga, .sup.72As
.sup.89Zr, and .sup.201Tl.
The monoclonal antibodies of the invention can also be labeled with a
paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic
resonance imaging (MRI) or electron spin resonance (ESR). In general, any
conventional method for visualizing diagnostic imaging can be utilized.
Usually gamma and positron emitting radioisotopes are used for camera
imaging and paramagnetic isotopes for MRI. Elements which are particularly
useful in such techniques include .sup.157Gd, .sup.55Mn, .sup.162Dy,
.sup.52Cr, and .sup.56Fe.
The monoclonal antibodies of the invention can be used in vitro and in vivo
to monitor the course of amelioration of a GDF-8-associated disease in a
subject. Thus, for example, by measuring the increase or decrease in the
number of cells expressing antigen comprising a polypeptide of the invention
or changes in the concentration of such antigen present in various body
fluids, it would be possible to determine whether a particular therapeutic
regimen aimed at ameliorating the GDF-8-associated disease is effective. The
term "ameliorate" denotes a lessening of the detrimental effect of the
GDF-8-associated disease in the subject receiving therapy.
The present invention identifies a nucleotide sequence that can be expressed
in an altered manner as compared to expression in a normal cell, therefore
it is possible to design appropriate therapeutic or diagnostic techniques
directed to this sequence. Thus, where a cell-proliferative disorder is
associated with the expression of GDF-8, nucleic acid sequences that
interfere with GDF-8 expression at the translational level can be used. This
approach utilizes, for example, antisense nucleic acid and ribozymes to
block translation of a specific GDF-8 mRNA, either by masking that mRNA with
an antisense nucleic acid or by cleaving it with a ribozyme. Such disorders
include neurodegenerative diseases, for example.
Antisense nucleic acids are DNA or RNA molecules that are complementary to
at least a portion of a specific mRNA molecule (Weintraub, Scientific
American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize
to the corresponding mRNA, forming a double-stranded molecule. The antisense
nucleic acids interfere with the translation of the mRNA, since the cell
will not translate a mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily synthesized and
are less likely to cause problems than larger molecules when introduced into
the target GDF-8-producing cell. The use of antisense methods to inhibit the
in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.
Biochem., 172:289, 1988).
Ribozymes are RNA molecules possessing the ability to specifically cleave
other single-stranded RNA in a manner analogous to DNA restriction
endonucleases. Through the modification of nucleotide sequences which encode
these RNAs, it is possible to engineer molecules that recognize specific
nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med.
Assn., 260:3030, 1988). A major advantage of this approach is that, because
they are sequence-specific, only mRNAs with particular sequences are
There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff,
Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes
recognize sequences which are four bases in length, while "hammerhead"-type
ribozymes recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence will
occur exclusively in the target mRNA species. Consequently, hammerhead-type
ribozymes are preferable to tetrahymena-type ribozymes for inactivating a
specific mRNA species and 18-based recognition sequences are preferable to
shorter recognition sequences.
The present invention also provides gene therapy for the; treatment of cell
proliferative or immunologic disorders which are mediated by GDF-8 protein.
Such therapy would achieve its therapeutic effect by introduction of the
GDF-8 antisense polynucleotide into cells having the proliferative disorder.
Delivery of antisense GDF-8 polynucleotide can be achieved using a
recombinant expression vector such as a chimeric virus or a colloidal
dispersion system. Especially preferred for therapeutic delivery of
antisense sequences is the use of targeted liposomes.
Various viral vectors which can be utilized for gene therapy as taught
herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA
virus such as a retrovirus. Preferably, the retroviral vector is a
derivative of a murine or avian retrovirus. Examples of retroviral vectors
in which a single foreign gene can be inserted include, but are net limited
to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number
of additional retroviral vectors can incorporate multiple genes. All of
these vectors can transfer or incorporate a gene for a selectable marker so
that transduced cells can be identified and generated. By inserting a GDF-8
sequence of interest into the viral vector, along with another gene which
encodes the ligand for a receptor on a specific target cell, for example,
the vector is now target specific. Retroviral vectors can be made target
specific by attaching, for example, a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target the
retroviral vector. Those of skill in the art will know of, or can readily
ascertain without undue experimentation, specific polynucleotide sequences
which can be inserted into the retroviral genome or attached to a viral
envelope to allow target specific delivery of the retroviral vector
containing the GDF-8 antisense polynucleotide.
Since recombinant retroviruses are defective, they require assistance in
order to produce infectious vector particles. This assistance can be
provided, for example, by using helper cell lines that contain plasmids
encoding all of the structural genes of the retrovirus under the control of
regulatory sequences within the LTR. These plasmids are missing a nucleotide
sequence which enables the packaging mechanism to recognize an RNA
transcript for encapsidation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .PSI.2, PA317 and PA12, for
example. These cell lines produce empty virions, since no genome is
packaged. If a retroviral vector is introduced into such cells in which the
packaging signal is intact, but the structural genes are replaced by other
genes of interest, the vector can be packaged and vector virion produced.
Alternatively, NIH 3T3 or other tissue culture cells can be directly
transfected with plasmids encoding the retroviral structural genes gag, pol
and env, by conventional calcium phosphate transfection. These cells are
then transfected with the vector plasmid containing the genes of interest.
The resulting cells release the retroviral vector into the culture medium.
Another targeted delivery system for GDF-8 antisense polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. The preferred colloidal system of this invention is a liposome.
Liposomes are artificial membrane vesicles which are useful as delivery
vehicles in vitro and in vivo. It has been shown that large unilamellar
vesicles (LUV), which range in size from 0.2-4.0 .mu.m can encapsulate a
substantial percentage of an aqueous buffer containing large macromolecules.
RNA, DNA and intact virions can be encapsulated within the aqueous interior
and be delivered to cells in a biologically active form (Fraley, et al.,
Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes
have been used for delivery of polynucleotides in plant, yeast and bacterial
cells. In order for a liposome to be an efficient gene transfer vehicle, the
following characteristics should be present: (1) encapsulation of the genes
of interest at high efficiency while not compromising their biological
activity; (2) preferential and substantial binding to a target cell in
comparison to non-target cells; (3) delivery of the aqueous contents of the
vesicle to the target cell cytoplasm at high efficiency; and (4) accurate
and effective expression of genetic information (Mannino, et al.,
Biotechniques, 6:682, 1988).
The composition of the liposome is usually a combination of phospholipids,
particularly high-phase-transition-temperature phospholipids, usually in
combination with sterols. especially cholesterol. Other phospholipids or
other lipids may also be used. The physical characteristics of liposomes
depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl
compounds, such as phosphatidylglycerol. phosphatidylcholine,
phosphatidylserine, phosphatidylethanclamine, sphingolipids, cerebrosides,
and gangliosides. Particularly useful are diacylphosphatidylglycerols, where
the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18
carbon atoms, and is saturated. Illustrative phospholipids include egg
phosphatidyl-choline, dipalmitoylphosphatidylcholine and
The targeting of liposomes can be classified based on anatomical and
mechanistic factors. Anatomical classification is based on the level of
selectivity, for example, organ-specific, cell-specific, and
organelle-specific. Mechanistic targeting can be distinguished based upon
whether it is passive or active. Passive targeting utilizes the natural
tendency of liposomes to distribute to cells of the reticuloendothelial
system (RES) in organs which contain sinusoidal capillaries. Active
targeting, on the other hand, involves alteration of the liposome by
coupling the liposome to a specific ligand such as a monoclonal antibody,
sugar, glycolipid, or protein, or by changing the composition or size of the
liposome in order to achieve targeting to organs and cell types other than
the naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of
ways. In the case of a liposomal targeted delivery system, lipid groups can
be incorporated into the lipid bilayer of the liposome in order to maintain
the targeting ligand in stable association with the liposomal bilayer.
Various linking groups can be used for joining the lipid chains to the
Due to the expression of GDF-8 in muscle and adipose tissue, there are a
variety of applications using the polypeptide, polynucleotide, and
antibodies of the invention, related to these tissues. Such applications
include treatment of cell proliferative disorders involving these and other
tissues, such as neural tissue. In addition, GDF-8 may be useful in various
gene therapy procedures.
The data in Example 6 (see Original Patent) shows that the human GDF-8 gene
is located on chromosome 2. By comparing the chromosomal location of GDF-8
with the map positions of various human disorders, it should be possible to
determine whether mutations in the GDF-8 gene are involved in the etiology
of human diseases. For example, an autosomal recessive form of juvenile
amyotrophic lateral sclerosis has been shown to map to chromosome 2 (Hentati,
et al., Neurology, 42 [Suppl.3]:201, 1992). More precise mapping of GDF-8
and analysis of DNA from these patients may indicate that GDF-8 is, in fact,
the gene affected in this disease. In addition, GDF-8 is useful for
distinguishing chromosome 2 from other chromosomes.
Claim 1 of 2 Claims
1. A method of treating a skeletal
musculodegenerative disease associated with expression of GDF-8 in a
subject, comprising contacting cells of the subject with a reagent which
suppresses the GDF-8 activity, wherein the reagent is an antibody that
binds the GDF-8 polypeptide of SEQ ID NO:12 or 14.
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