AU2002364946B2 - Bone morphogenic proteins (BMP), BMP receptors and BMP binding proteins and their use in the diagnosis and treatment of glaucoma - Google Patents

Description

BONE MORPHOGENIC PROTEINS (BMP), BMP RECEPTORS AND BMP

BINDING PROTEINS AND THEIR USE IN THE DIAGNOSIS AND

TREATMENT OF GLAUCOMA

1. Field of the Invention

The present invention discloses methods and reagents for diagnosing and treating glaucoma and related disorders.

2. Description of the Related Art

"Glaucomas" are a group of debilitating eye diseases that are the leading cause of irreversible blindness in the United States and other developed nations. Primary Open Angle Glaucoma ("POAG"), the most common form of glaucoma, is characterized by the degeneration of the trabecular meshwork, leading to obstruction of the normal ability of aqueous humor to leave the eye without closure of the space (e.g., the "angle") between the iris and cornea (Vaughan, D. et al, (1992)). A characteristic of such obstruction in this

disease is an increased intraocular pressure ("IOP"), resulting in progressive visual loss and blindness if not treated appropriately and in a timely fashion.

The disease is estimated to affect between 0.4% and 3.3% of all adults over 40 years old

(Leske, M. C. et al. (1986); Bengtsson, B. (1989); Strong, N. P. (1992)). Moreover, the

prevalence of the disease rises with age to over 6% of those 75 years or older (Strong, N. P., (1992)).

Because increased IOP is a readily measurable characteristic of glaucoma, the

diagnosis of the disease is largely screened for by measuring intraocular pressure

(tonometry) (Strong, N. P. (1992); Greve, M. et al. (1993)). Unfortunately, because

glaucomatous and normal pressure ranges overlap, such methods are of limited value unless multiple readings are obtained (Hitchings, R. A., (1993); Tuck, M. W. et al. (1993); Vaughan, D. et al, (1992); Vernon, S. A., (1993)). For this reason, additional methods,

such as direct examination of the optic disk and determination of the extent of a patient's

visual field loss are often conducted to improve the accuracy of diagnosis (Greve, M. et

al, (1993)).

Glaucoma affects three separate tissues in the eye. The elevated IOP associated

with POAG is due to morphological and biochemical changes in the trabecular meshwork

(TM), a tissue located at the angle between the cornea and iris. Most of the nutritive

aqueous humor exits the anterior segment of the eye through the TM. The progressive loss of TM cells and the build-up of extracellular debris in the TM of glaucomatous eyes leads to increased resistance to aqueous outflow (Lutjen-Drecoll and Rohen 1996; Rohen 1983;

Rohen et al 1993; Grierson and Calthorpe 1988), thereby raising IOP. Elevated IOP, as

well as other factors such as ischemia, cause degenerative changes in the optic nerve head (ONH) leading to progressive "cupping" of the ONH (Varma and Minckler 1996;

Hernandez and Gong 1996; Hernandez et al 1990; Hernandez and Pena 1997; Morrison et al. 1990) and loss of retinal ganglion cells (Quigley et al. 2000; Quigley 1999; Quigley et

al. 1995; Kerrigan et al. 1997) and axons. The detailed molecular mechanisms responsible

for glaucomatous damage to the TM, ONH, and the retinal ganglion cells are unknown.

Current glaucoma therapy is directed to lowering IOP, a major risk factor for the

development and progression of glaucoma. These therapies lower IOP, but they do not

directly address the pathogenic mechanisms, and the disease continues to progress. At

least half of patients with glaucoma are undiagnosed, and by the time patients are

diagnosed with glaucoma, they have already lost approximately 40% of their retinal ganglion cells. Therefore, methods for earlier detection and diagnosis of glaucoma are

needed.

In view of the importance of glaucoma, and the at least partial inadequacies of prior

methods of diagnosis, it would be desirable to have an improved, more accurate method

for diagnosing glaucoma in its early stages. In addition, it would be desirable to have new

therapeutic agents that address glaucomatous pathogenic mechanisms.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of the prior art by providing methods and kits for the early diagnosis of glaucoma, for treating glaucoma, and

for the identification of compounds useful in the treatment of glaucoma.

In certain specific embodiments, the invention provides a method for diagnosing

glaucoma in a sample obtained from a cell or bodily fluid by detecting altered expression

of a bone morphogenic protein family member gene. The method generally includes the

steps of: a) obtaining a tissue or fluid sample from a patient suspected of having

glaucoma;

b) extracting DNA from said sample;

c) obtaining a plurality of PCR primers, wherein said primers each comprise a

sequence consisting of from 18 to 1547 contiguous nucleotides from SEQ

ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 37,

SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID

NO:47, or SEQ ID NO:53; d) amplifying regions of the extracted DNA using said primers to obtain a

PCR product; e) resolving the PCR product; and

f) identifying differences between the sequence of the PCR product and the

normal gene sequence;

where a difference between the amplified sequence and the normal gene sequence

is diagnostic of glaucoma.

In general, the methods of the invention may include obtaining a sample from an

individual and extracting DNA from said sample. Select PCR primers for specific members of the BMP gene family are then used to amplify relevant regions of the

extracted gene to obtain a PCR product. The PCR product is resolved by a technique that

effectively identifies DNA sequence differences between the normal and mutated form of

the specific BMP family gene being evaluated (the extracted DNA). Identified differences between the sequences are indicative of glaucoma.

The tissue or fluid sample for use in the methods of the invention may be blood or

buccal cells. Typically, the primer sequences will have a length of between about 10, 15 or 18

nucleotides to about 20, or to about 30 nucleotides. Longer sequences, e.g., 40, 50, 80, 90,

95, 100, even up to full length, are even more preferred for certain embodiments. Lengths

of oligonucleotides of at least about 18 to 20 nucleotides are well accepted by those of skill

in the art as sufficient to allow sufficiently specific hybridization so as to be useful as a

molecular probe, as described by Lathe (1985), which reference is specifically

incorporated herein by reference for this purpose. Preferably, the nucleotide sequence will

consist of from 20 to 100 contiguous nucleotides of SEQ ID NO:l, SEQ ID NO:3, SEQ ID

NO:5, SEQ ID NO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ fD NO:47, or SEQ ID NO:53. It is also contemplated that the primer sequences may consist of sequences of at least 10, 15 or 18 contiguous nucleotides from

the sequences of BMP receptor genes and from BMP-associated proteins, the sequences of

which are known.

Nucleic acid molecules having stretches of 10, 18, 20, 30, 50, 60, 65 or even up to

and including 100 nucleotides or so, complementary to any one of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,

SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:53, have utility as hybridization probes. Primers or probes having a nucleotide length of about 18

nucleotides are recognized by those of skill in the art to provide highly specific

hybridization to a target sequence. The total size of the fragment, as well as the size of the complementary stretches, will ultimately depend on the intended use of application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization

embodiments, wherein the length of the complementary region may be varied, such as

between about 10, 18, 20 or 30 and about 50, 60, 70, 80, 80 or 100 nucleotides, or even full length according to the complementary sequences one wishes to detect.

In specifically preferred embodiments, the primers will consist of contiguous

sequences from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID

NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47,

or SEQ ED NO:53. In other preferred embodiments, the primers will consist of

contiguous sequences from BMP receptor genes (disclosed in ten Dijke et al. 1993;

Astrom et al. 1999; Nohno et al. 1995, all incorporated herein by reference) or from BMP-

associated genes, such as chordin (NCBI NM 029130), gremlin (Murphy et al. 1999;

McMahon et al 2000), follistatin (NCBI NM_003892) or bambi (NCBI NM_005791). Most preferably, the primers will consist of contiguous sequence from SEQ ID NO:3. In certain aspects, at least some of the primers may further include a detectable label.

In other embodiments, the invention provides a method for treating glaucoma by

administering to a patient in need thereof a composition comprising a sequence consisting

of at least one compound selected from the group consisting of a BMP2 agonist, a BMP4

agonist, a BMP5 agonist, a BMP7 agonist, a Smad 1/5 agonist, a chordin antagonist, a

gremlin antagonist and a follistatin antagonist.

In additional aspects, the present invention provides a method for identifying a

therapeutic agent for the treatment of glaucoma. Therapeutic agents may be identified, for

example, by: a) obtaining a first composition comprising a population of recombinant cells expressing BMP-2A, BMP4, BMP-5, or BMP7;

b) obtaining a candidate substance; c) incubating said composition and said candidate substance;

testing said composition for its ability to turn on BMP-induced Smad signaling pathways

and/or BMP-regulated gene expression; and identifying a candidate substance that inhibits,

or stimulates, these downstream effects of BMP.

Another aspect of the invention are diagnostic kits containing sequences of the

present invention and suitable reagents such as a detectable label linked to a protein,

peptide or the antibody itself. Alternatively, the detectable label may be linked to a second

sequence which selectively hybridizes to a sequence of the invention.

Related embodiments include therapeutic kits which include pharmaceutically-

acceptable formulations of either the nucleic acid sequences or peptide or protein sequences disclosed herein. Such kits are useful in the detection of altered expression of

the BMP genes and proteins in clinical samples for the diagnosis of glaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings form part of the present specification and are included to further

demonstrate certain aspects of the present invention. The invention may be better

understood by reference to one or more of these drawings in combination with the detailed

description of specific embodiments presented herein.

FIG. 1. Nucleotide and amino acid sequence of BMP2A.

FIG.2. Nucleotide and amino acid sequence of BMP4. FIG. 3. Nucleotide and amino acid sequence of BMP5.

FIG. 4. Nucleotide and amino acid sequence of BMP7.

FIG. 5. Bone morphogenic protein signaling pathway. Bone Morphogenic Protein (BMP)

dimers bind to a membrane complex composed of BMP receptors 1 and 2, which are serin/threonine kinases. The regulatory Smads (Smadl/Smad5) become phosphorylated

and associate with a co-Smad (Smad 4). This resulting Smad complex enters the nucleus

where it associates with transcription factors (TF) and regulates gene expression. BMP

associated proteins act as BMP antagonists by binding BMPs and preventing BMP

interaction with BMP receptors.

FIG. 6. BMP expression in human TM cells and tissues. Ethidium bromide-stained

agarose gel of BMP PCR products from cDNA samples generated from RT-PCR analysis

of BMP expression in human TM cells (lanes 1-5) and tissues (lanes 6-7). L = base pair

markers. C = PCR negative control lane, β-actin was used as a positive RT-PCR internal

control. FIG. 7. BMP receptor expression in human TM cells and tissues. Ethidium bromide-

stained agarose gel of PCR products from cDNA samples generated from RT-PCR analysis of BMP receptor expression in human TM cells (lanes 1-5) and tissues (lanes 6-

7). L = base pair markers. C = PCR negative control lane, β-actin was used as a positive

RT-PCR internal control.

FIG. 8. BMP expression in human ONH astrocytes, ONH tissues, and human brain

astrocytes. Ethidium bromide-stained agarose gel of PCR products from cDNA samples generated from RT-PCR analysis of BMP expression in human ONH astrocyte (lanes 1-5),

ONH tissue (lane 6), and human brain astrocytes (lane 7). L = base pair markers. C =

PCR negative control lane, β-actin was used as a positive RT-PCR internal control.

FIG. 9. BMP expression in human lamina cribrosa cell lines. Ethidium bromide-stained agarose gel of PCR products from cDNA samples generated from RT-PCR analysis of

human lamina cribrosa cells (lanes 1-9). L = base pair markers. C = PCR negative control

lane, β-actin was used as a positive RT-PCR internal control.

FIG. 10. BMP receptor expression in human ONH astrocytes,ONH tissues, and human

brain astrocytes. Ethidium bromide-stained agarose gel of PCR products from cDNA

samples generated from RT-PCR analysis of BMP receptor expression in human optic

nerve head astrocytes (ONA) (lanes 1-5), ONH tissue (lane 6), and human brain astrocytes

(lane 7). L = base pair markers. C = PCR negative control lane, β-actin was used as a

positive RT-PCR control.

FIG. 11. BMP receptor expression in human lamina cribrosa cell lines. Ethidium

bromide-stained agarose gel of PCR products from cDNA samples generated from RT-

PCR analysis of human lamina cribrosa cells (lanes 1-9). L = base pair markers. C = PCR

negative control lane, β-actin was used as a positive RT-PCR control. FIG. 12. Western immunoblot of BMP and BMP receptor expression in cultured human

TM cells, optic nerve head astrocytes (ONA), and lamina cribrosa cells.

Chemiluminescent detection of BMP proteins and BMP receptors in human trabecular

meshwork cells (lanes 1-2), ONH astrocytes (lanes 3-4), and lamina cribrosa cells (lanes 5-

6). Protein size indicated in kDa.

FIG. 13. BMP associated protein mRNA expression in human TM cells. Ethidium

bromide-stained agarose gel of PCR products from cDNA samples generated from RT-

PCR analysis of human TM cells (lanes 1-5). L = base pair markers. C = PCR negative

control lane, β-actin was used as a positive RT-PCR internal control.

FIG. 14. BMP associated protein mRNA expression in human lamina cribrosa cells and

ONH astrocytes. Ethidium bromide-stained agarose gel of PCR products from cDNA

samples generated from RT-PCR analysis of lamina cribrosa (LC) cells (lanes 1-7) and

ONH astrocytes (ONA) (lanes 8-1 1). L = base pair markers. C = PCR negative control

lane, β-actin was used as a positive RT-PCR internal control.

FIG. 15. Illustrates increased expression of the BMP antagonist gremlin (CKTSFIBI) in

glaucomatous TM cells. Gene expression was assessed using Affymetrix gene arrays

(Affymetrix gene chip U133A).

DETAILED DESCRIPTION PREFERRED EMBODIMENTS

The trabecular meshwork has been proposed to play an important role in the

normal flow of the aqueous humor, and has been presumed to be the major site of outflow

resistance in glaucomatous eyes. Human trabecular meshwork (HTM) cells are

specialized cells which line the outflow channels by which aqueous humor exits the eye. Altered synthetic function of the cells may be involved in the pathogenesis of POAG,

steroid glaucoma, and other types of glaucoma.

Despite years of intensive research, the precise molecular mechanisms responsible

for glaucomatous damage to the eye are not known. Recent research has suggested that

growth factors may be important in maintaining normal homeostasis in the ocular tissues

associated with glaucoma, and alterations in growth factor/growth factor receptors may

play a role in glaucoma pathogenesis. Growth factors area very large family of

polypeptides that control cell growth and differentiation. These molecules have a variety of cell-specific effects on gene expression, extracellular matrix composition and

deposition, cytoskeletal organization, and regulation of cellular functions. The TM expresses a wide variety of growth factors, growth factor receptors (Tripathi et al 1993a;

Tripathi et al 1993b; Tripathi et al 1994a; Tripathi et al. 1994b; Wordinger et al. 1998;

Wordinger et al. 1999) as well as neurotrophin/neurotrophic factors and their receptors

(Liu et al. 2001; Wordinger et al. 2000). ONH astrocytes and lamina cribrosa cells, two

cell types of the optic nerve head, express growth factors, neurotrophins and their receptors

(Lambert et al 2001; Pena et al. 1999). The aqueous humor also contains a variety of

growth factors including FGF2, EGF, TGFβ, HGF (Tripathi et al 1996; Tripathi et al.

1991; Tripathi et al 1992; Hu and Ritch 2001) as well as neurotrophins (Chundru et al.

2000). Elevated levels of aqueous humor TGFβ-2 and HGF have been reported in POAG

patients (Tripathi et al 1994c; Inatani et al 2001; Picht et al. 2001). Growth factors may

be involved in glaucoma by altering the normal development and/or function of the TM

and ONH.

The present invention stems in part from the recognition that bone morphogenic

proteins (BMPs) not only induce bone and cartilage formation but are multifunctional cytokines having a wide range of effects on numerous cell types (Hogan 1996; Reddi 1997) and are expressed by both human trabecular meshwork (HTM) and optic nerve head

(ONH) cells (Wordinger et al 2002). BMPs are members of the TGFβ superfamily, and

there are approximately 15-20 BMPs genes in man, 3 BMP receptors, and a number of

BMP associated proteins that function as BMP antagonists (Yamashita et al. 1996). BMPs

signal via a receptor complex consisting of BMPR-I and BMPR-IJ. It has been reported

that superfamily members TGFβ and TGFβR (Agarwal et al. 1997; Lambert et al. 1997)

and GDNF and GDNFR (Wordinger et al. 1999; Liu et al. 1999) are expressed by both

HTM and ONH cells. BMPs and BMP receptors are expressed in ocular tissues (Obata et al. 1999; You

et al. 1999), but previous reports have focused on ocular development. The function of BMPs is important in ocular development since targeted disruption of genes encoding

BMPs in mice leads to severe developmental defects in the retina and the lens (Jena et al. 1997; Luo et al. 1995; Dudley et al. 1995). BMP-2, BMP-4 and BMP-7 are involved in

the development of the lens and retina (Jena et al. 1997; Furuta and Hogan 1998; Reddi 2000; Trousse et al. 2001). BMP-6 and BMP-7 also appear to play a role in protecting

neurons from hypoglycemic or ischemic damage (Nonner et al. 2001; Liu et al. 2001), and

BMP2 has been shown to enhance ganglion cell neurotrophin expression (Zhang et al.

1998). Heterozygous knock-out mice haploinsufficient for Bmp4 have ocular phenotypes

including anterior segment dysgenesis, elevated IOP, and optic nerve abnormalities (Chang

et al. 2001). There has been very limited information published concerning the role of

BMPs in the human postnatal eye.

Mohan and colleagues (1998) reported that BMP-2 and BMP-4 and BMP receptors

were expressed in cells of the adult cornea and suggested that BMP function might include corneal keratocyte proliferation and apoptosis. You and colleagues (1999) verified this

study and also reported the expression of BMP-3, BMP-5, and BMP-7 in ex vivo and

cultured corneal epithelium and stromal cells. They reported that the level of BMP

transcription was higher in the stroma while the level for receptors was higher in cultured

corneal epithelial cells.

Using RT-PCR, the present inventors discovered mRNAs for BMPs, BMP

receptors BMPR-IA, BMPR-IB and BMPR-IJ, as well as BMP binding proteins gremlin,

chordin, follistatin, and bambi, in HTM, lamina cribosa (LC) and ONH astrocyte cell lines

and tissues (Wordinger et al. 2002). The present inventors further discovered that HTM

and ONH cells express proteins BMP-2, BMP-4, BMP-5 and BMP-7.

Glaucoma will be diagnosed by characterization of genetic changes in genes of

members of the BMP signaling family. As used herein, the phrases "bone morphogenic

protein family member gene" and "BMP signaling family" refer to all BMPs, BMP

receptors and associated proteins. The term "genetic changes" is well known by those

skilled in the art. There are numerous examples of diseases associated with genetic

changes in specific genes (for examples see Cummings 1997; Strachan, et al. 1996; Jorde,

et al. 1999). Genetic changes in a specific gene (e.g. BMP) can be determined using a

variety of techniques well known by those skilled in the art, such as: SSCP, DGGE, ASO,

RFLP, heteroduplex analysis, CCM, PTT, and RNase cleavage (see Birren, et al. 1998).

Glaucoma may be caused by altered expression of one or more BMP family genes

in the eye, which leads to elevated IOP and/or glaucomatous optic neuropathy. "Altered

BMP gene expression" means expression of this gene product that is different from

normal. The term may also refer to alterations in the sequence of the gene or protein. The

normal BMP gene has been well characterized (see above), and the expression of BMP has been reported in a variety of tissues including the TM and ONH. Genetic changes in the

coding region of BMP family genes may alter the function of these proteins. Genetic

changes outside the coding region may also lead to glaucoma.

It is well known by those skilled in the art that "changes outside" of the coding

region of a specific gene are important in the regulation of gene expression. For example,

the region upstream (5') of the coding region of most genes is known as the promoter

region which "promotes" and regulates the expression of that gene. The promoter region

contains numerous nucleotide sequences recognized by various transcription factors and

DNA binding proteins that are responsible for activation or repression of gene expression.

Regions downstream (3') of the gene can determine poly-adenylation of the gene product, thereby regulating RNA processing and translation of the gene product.

The altered expression of BMP genes or mutations in the sequence of the genes

that is indicative of glaucoma may be detected using techniques well known to those of skill in the art. For example, it is contemplated that a nucleic acid fragment of almost any

length may be employed, with the total length preferably being limited by the ease of

preparation and use in the intended protocol. The nucleic acid sequences disclosed herein

may also have utility as probes or primers in nucleic acid hybridization embodiments. As

such, it is contemplated that nucleic acid segments that comprise a sequence region that

consists of at least a 14 nucleotide long contiguous sequence that has the same sequence

as, or is complementary to, a 14 nucleotide long contiguous sequence of BMP-2 A (SEQ

ID NO:l), BMP-4 (SEQ ID NO:3), BMP-5 (SEQ ID NO:5), BMP-7 (SEQ ID NO:7),

BMP-RIA (SEQ ID NO:37), BMP-RIB (SEQ ID NO:39), BMP-P I (SEQ ID NO:41),

chordin (SEQ ID NO:43), gremlin (SEQ ID NO:45), follistatin (SEQ ID NO:47), or bambi

(SEQ ID NO:53) will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 nucleotides (including all intermediate lengths), and even up to full length sequences of

about 1547 nucleotides (for BMP-2A), 1946 nucleotides (for BMP-4), 2153 nucleotides

(for BMP-5) and 1878 nucleotides (for BMP-7), 2932 nucleotides (for BMP-RIA), 2032

nucleotides (for BMP-RJJ3), 3611 nucleotides (for BMP-RII), 3561 nucleotides (for chordin), 4049 nucleotides (for gremlin), 1386 nucleotides (for follistatin), and 1523

nucleotides (for bambi) will also be of use in certain embodiments.

It will be readily understood that "intermediate lengths", in this context, means any

length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;

including all integers through the 200-500; 500-1,000; 1,000-2,000 ranges, up to and

including sequences of 2,001, 2002, 2050, 2051, and the like.

The ability of such nucleic acid probes and primers to specifically hybridize to

BMP coding sequences and primers to specifically amplify BMP sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample.

However, other uses are envisioned, including the use of the sequence information for the

preparation of mutant species primers, or primers for use in preparing other genetic

constructions.

Nucleic acid molecules having sequence regions consisting of contiguous

nucleotide stretches of 10, 20, 30, 50, or even of 100-200 nucleotides or so, identical or

complementary to BMP-2A (SEQ ID NO:l), BMP4 (SEQ ID NO:3), BMP-5 (SEQ ID

NO:5), BMP7 (SEQ ID NO:7) , BMP-RIA (SEQ ID NO:37), BMP-RIB (SEQ ID NO:39),

BMP-Rπ (SEQ ID NO:41), chordin (SEQ ID NO:43), gremlin (SEQ ID NO:45), follistatin

(SEQ ID NO:47), or bambi (SEQ ID NO:53) are particularly contemplated as hybridization probes for use in, e.g., SNP evaluation and solid phase hybridization assays, in addition to Southern and northern blotting. This would allow BMP structural or

regulatory genes to be analyzed, both in tissues and cells. The total size of fragment, as

well as the size of the complementary stretch(es), will ultimately depend on the intended

use of application of the particular nucleic acid segment. Smaller fragments will generally

find use in hybridization embodiments, wherein the length of the contiguous

complementary region may be varied, such as between about 10 and about 100

nucleotides, but larger contiguous complementary stretches of up to about 1547

nucleotides (for BMP-2 A), 1946 nucleotides (for BMP-4), 2153 nucleotides (for BMP-5)

and 1878 nucleotides (for BMP-7), 2932 nucleotides (for BMP-RIA), 2032 nucleotides

(for BMP-PJB), 3611 nucleotides (for BMP-RH), 3561 nucleotides (for chordin), 4049 nucleotides (for gremlin), 1386 nucleotides (for follistatin), and 1523 nucleotides (for

bambi) may be used, according to the length complementary sequences one wishes to

detect. The use of a hybridization probe of about 10-14 nucleotides in length allows the

formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 10 bases in length are

generally preferred, though, in order to increase stability and selectivity of the hybrid, and

thereby improve the quality and degree of specific hybrid molecules obtained, one will

generally prefer to design nucleic acid molecules having gene-complementary stretches of

15 to 20 contiguous nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences

disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:l,

SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:53 and to select

any continuous portion of the sequence, from about 10 nucleotides in length up to and

including the full length sequence, that one wishes to utilize as a probe or primer. The

choice of probe and primer sequences may be governed by various factors, such as, by way

of example only, one may wish to employ primers from towards the termini of the total

sequence, or from the ends of the functional domain-encoding sequences, in order to

amplify further DNA.

The process of selecting, and preparing a nucleic acid segment that includes a contiguous sequence from within BMP-2A (SEQ ID NO:l), BMP4 (SEQ ID NO:3), BMP-

5 (SEQ ID NO:5), BMP7 (SEQ ID NO:7) , BMP-RIA (SEQ ID NO:37), BMP-RIB (SEQ

ID NO:39), BMP-RII (SEQ ID NO:41), chordin (SEQ ID NO:43), gremlin (SEQ ID NO:45), follistatin (SEQ ID NO:47), or bambi (SEQ ID NO:53) may alternatively be

described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme

digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced

using an automated oligonucleotide synthesizer. Also, fragments may be obtained by

application of nucleic acid reproduction technology, such as the PCR™ technology of U.S.

Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195 (each incorporated herein by reference), by

introducing selected sequences into recombinant vectors for recombinant production, and

by other recombinant DNA techniques generally known to those of skill in the art of

molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of BMP genes or cDNAs. Depending on the application envisioned, one will desire to employ varying

degrees of selectivity of hybridization to achieve varying degrees of selectivity of probe

towards target sequence. For applications requiring high selectivity, one will typically

desire to employ relatively stringent conditions to form the hybrids, e.g., one will select

relatively low salt and/or high temperature conditions, such as provided by 0.02M-0.15M

NaCl at temperatures of 50° C to 70° C. Such selective conditions tolerate little, if any,

mismatch between the probe and the template or target strand, and would be particularly suitable for examining BMP genes.

Of course, for some applications, for example, where one desires to prepare or identify mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate BMP encoding sequences from related species, functional

equivalents, or the like, less stringent hybridization conditions will typically be needed in

order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such a 0.15M-1.0M salt, at temperatures ranging from 20° C to 55° C.

Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hycbridizations. In any case, it is generally appreciated that

conditions can be rendered more stringent by decreasing NaCl concentrations or by the

addition of increasing amounts of formamide, which serves to destabilize the hybrid

duplex in the same manner as increased temperature. Thus, hybridization conditions can

be readily manipulated, and thus will generally be a method of choice depending on the

desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences

of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments,

one will likely desire to employ a fluorescent label or an enzyme tag, such as urease,

alkaline phosphatase or peroxidase, instead of radioactive or other environmental

undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are

known that can be employed to provide a means visible to the human eye or

spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be

useful both as reagents in solution hybridization as well as in embodiments employing a

solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic

acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on

the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following

washing of the hybridized surface so as to remove nonspecifically bound probe molecules,

specific hybridization is detected, or even quantified, by means of the label.

It will also be understood that this invention is not limited to the particular nucleic

acid and amino acid sequences of BMP-2A (SEQ ID NO:l), BMP4 (SEQ ID NO:3),

BMP-5 (SEQ ID NO:5), BMP7 (SEQ ID NO:7) , BMP-RIA (SEQ ID NO:37), BMP-PJB

(SEQ ID NO:39), BMP-RII (SEQ ID NO:41), chordin (SEQ ID NO:43), gremlin (SEQ ID

NO:45), follistatin (SEQ ID NO:47), or bambi (SEQ ID NO:53). Recombinant vectors

and isolated DNA segments may therefore variously include the BMP coding regions themselves, upstream or downstream regions of the genes, coding regions bearing selected

alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include BMP coding regions or may encode biologically

functional equivalent proteins or polypeptides that have variant amino acid sequences.

The DNA segments of the present invention encompass biologically functional

equivalent BMP proteins and polypeptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within

nucleic acid sequences and the proteins thus encoded. Alternatively, functionally

equivalent proteins or polypeptides may be created via the application of recombinant

DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by

man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test BMP mutants in order to examine binding activity at the molecular level.

The therapeutic agent for the treatment of glaucoma can be: a peptide or protein, a

peptide mimetic, an oligonucleotide or derivatized oligonucleotide, or small drug-like

molecule, all which affect one or more aspects of the ocular BMP pathways. Preferred

therapeutic agents are those that are: (1) BMP2, BMP4, BMP5, or BMP7 agonists; (2)

chordin, gremlin, follistatin, or bambi antagonists; and/or (3) Smadl, Smad5 and/or

Smad4 agonists.

The agent may be delivered directly to the eye (for example: topical ocular drops or

ointments; slow release devices in the cul-de-sac or implanted adjacent to the sclera or

within the eye; periocular, conjunctival, sub-Tenons, intracameral or intravitreal

injections) or parenterally (for example: orally; intravenous, subcutaneous or intramuscular injections; dermal delivery; etc.) using techniques well known by those skilled in the art.

The following are examples of possible formulations embodied by this invention.

(a) Topical ocular formulation wt. %

Agent that increases ocular BMP-4 expression 0.01 - 2

HPMC 0.5

Sodium chloride 0.8

BAC 0.01%

EDTA 0.01

NaOH/HCl qs pH 7.4

Purified water qs 100 mL

(b) Topical ocular formulation wt. %

Gremlin antagonist 0.01 - ^■ 2 HPMC 0.5

Sodium chloride 0.8 BAC 0.01 EDTA 0.01

NaOH/HCl qs pH 7.4 Purified water qs 100 mL

(c) Topical ocular formulation wt. %

Smad 1/5 agonist 0.01 - 2

HPMC 0.5

Sodium chloride 0.8

BAC 0.01

EDTA 0.01 NaOH/HCl qs pH 7.2

Purified water qs 100 mL

It is further contemplated that the compounds of the invention could be formulated

in intraocular insert devices.

A. Assay for therapeutic agents This invention is also useful for the discovery of new anti-glaucoma therapeutic

agents that are involved in the BMP signaling pathway (see FIG. 5). Selective BMP

ligands bind to BMP type I and type π serine/threonine kinase receptors (BMP-RI and BMP-RJJ) and transduce signal via Smad proteins. The BMP signal is propagated by

Smads through protein-protein and protein-DNA interactions (Attisano and Tuen Lee-

Hoefiich 2001). Regulatory Smad 1 and Smad 5 are activated (via phosphorylation) by

ligand bound BMP receptors (von Bubnoff and Cho 2001). These regulatory Smads then

interact with Smad 4 to form a heteromeric complex that translocates to the nucleus. This

complex is able to activate or repress the transcription of selective genes that recognize

this transcriptional complex, depending on which nuclear co-factors are present.

The BMP/Smad signaling pathway is negatively regulated by several mechanisms.

Certain BMP-binding proteins (such as gremlin, BAMBI, or follistatin) bind BMPs and inhibit their interaction with BMP receptors. In addition, there are inhibitory Smad

proteins (e.g. Smad 6 and Smad 7), which bind and inactivate BMP receptors. (Kowabata

et al. 1998; Itoh et al. 2000; Miyazono 2000). The present inventors have discovered that human TM cells, ONH astrocytes and lamina cribrosa cells express message and protein for the BMP receptor complex. Thus, these cells could respond to endogenous BMP .

ligands.

Various methods may be used to discover new anti-glaucoma therapeutic agents,

and these techniques are well known to those skilled in the art. For example, peptide or

peptide mimetic agents that act as agonists or inhibitors of BMPs can be discovered through molecular modeling of BMP/BMP receptor structures (Nickel et al. 2001). BMP

signal fransduction involves select sets of Smad proteins (Kawabata et al. 1998; Itoh et al.

2000; Attiseno et al. 2000). Select BMP agonists and Smad agonists can be discovered

using cell based assays. The test cell should express the appropriate BMP receptor(s) and

possess the appropriate BMP signaling pathway. Because one of the major effects of BMP

signaling is the alteration of gene expression, BMP agonists and Smad agonists can be discovered by screening for BMP-induced genes. The induction of BMP regulated genes also may be assayed by quantitating levels of mRNA using quantitative RT-PCR (Wang et

al. 2001), DNA microarrays, or reporter gene constructs. There are natural inhibitors of

BMP signaling, the BMP binding proteins (also known as BMP-associated proteins), such

as chordin, gremlin, and follistatin. Antagonists of the protein inhibitors can be discovered

using ligand binding assays. For example, test agents can be added to recombinant

purified gremlin, and those agents that bind to gremlin are identified using a variety of

techniques known to those skilled in the art. To determine whether these agents are gremlin antagonists, a cell based assay similar to that described above is used.

It is contemplated that any known in vitro and in vivo screening models may be used in conjunction with the present invention to identify new glaucoma therapies directed

to the BMP family of genes. Such models are well known to those skilled in the art and

their practice has become routine. Small peptides or peptide mimetics can be designed based on structure/function knowledge of the BMP, BMPR, and/or BMP binding protein

gene products. Ligand binding assays can be used to detect small molecules that bind to BMPs, BMPRs, or BMP binding proteins. Cell based assays can look at the effects of

various agents on BMP signaling pathways. Knock-in cell lines containing BMP family

gene promoters coupled to a reporter gene can be generated to look for agents that alter

BMP family member gene expression. These assays can be used to identify both agonist

and antagonist molecules. Ex vivo assays, such as perfusion cultured anterior segments

from human eyes (Clark et al. 1995a; Pang et al. 2000), can be used to examine the effects

of agents on IOP and on BMP signaling in TM tissue. Rodent models of glaucoma can be

generated using well-known techniques to create stable BMP family member transgenic,

knockout, or knock-in strains of mice and rats. These rodent models can be used to screen for agents that alter the glaucoma-like phenotype(s) (e.g. tonometry to evaluate effects on

IOP, histology to evaluate effects on glaucomatous optic neurology).

B. Kits The present invention provides methods, compositions and kits for the early

detection of glaucoma. The kits can contain a nucleic acid segment encoding a BMP

polypeptide or protein. The kit can further contain reagents for detecting an interaction

between a sample and a nucleic acid or peptide of the present invention. The provided

reagent can be radio-, fluorescently- or enzymatically-labeled. The kit can contain a

known radiolabeled agent capable of binding or interacting with a nucleic acid or peptide or protein of the present invention.

The reagent of the kit can be provided as a liquid solution, attached to a solid

support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is

attached to a solid support, the solid support can be chromatography media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry

powder, the powder can be reconstituted by the addition of a suitable solvent, that may be

provided.

In still further embodiments, the present invention concerns diagnostic methods

and associated kits for the diagnosis of glaucoma. It is proposed that the BMP associated

peptides and nucleic acids of the invention may be employed to detect polymorphisms or

mutations in the BMP nucleic acids from patient samples. In general, these methods will

include first obtaining a sample suspected of containing such a polymorphism or mutation,

contacting the sample with a peptide or nucleic acid of the present invention, as the case may be, under conditions effective to allow the formation of a complex, and then detecting

the presence of the complex.

In general, the detection of complex formation is quite well known in the art and

may be achieved through the application of numerous approaches. For example, the

present invention contemplates the application of ELISA, RIA, indirect fluorescence

techniques and the like. Generally, complex formation will be detected through the use of

a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of

a secondary binding ligand.

The following examples are representative of the techniques employed by the

inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the

invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope

of the invention.

Example 1 Cell culture: Human TM cells and ONH cells were generated from donor eyes as

described (Steely et al. 1992; Steely et al. 2000; Wilson et al. 1993; Clark et al. 1994;

Clark et al. 1995b; Clark et al. 1995c; Clark et al 1996; Clark et al. 2001a; Clark et al

2001b; Dickerson et al 1998; Wordinger et al 1998; Wordinger et al. 1999; Wordinger et

al. 2000; Wordinger et al. 2002; Lambert et al. 2001; Agarwal et al. 1999; Liu et al.

2001). TM cells were grown from TM explants of donors ranging in age from 6 days to 90

years. Human optic nerve head astrocytes and lamina cribrosa (LC) cells were generated from carefully dissected optic nerve heads (donors aged 2 days to 90 years) and characterized according to previous reports (Lambert et al. 2001; Clark et al. 1995a). The

cells wers grown to confluency in the following media: Ham's F10 media (JRH Biosciences, Lenexa, KS) containing 10% fetal bovine serum (HyClone, Logan, UT) and

antibiotics (Gibco BRL-Life Technologies, Grand Island, NY) for TM cells; Dulbecco's

modified Eagle's media (DMEM, HyClone) containing 10% FBS for LC cells; and

astrocyte growth medium (AGM, Clonetics, San Diego, CA) containing 5% FBS for ONH

astrocytes.

RT-PCR: Human TM and ONH tissues also were dissected from donor eyes (Wordinger et al. 1998; Wang et al. 2001). Total RNA was extracted from the TM and

ONH cells and tissues using TRIzol extraction (Gibco BRL-Life Technologies), and

cDNA prepared by reverse transcription using standard procedures (Wordinger et al. 1998; Wordinger et al. 1999; Wordinger et al 2000; Wordinger et al. 2002). PCR primers were designed using the Oligos 4.0 software program (see primer pairs in Table 1). All primer

pairs were designed so that amplification of potentially contaminated genomic DNA

sequences would produce mRNA PCR products that would be substantially larger than expected, because intron sequences that were excised during RNA processing would be

included in genomic DNA. The β-actin PCR primers,

AGGCCAACCGCGAGAAGATGACC (upstream) and

GAAGTCCAGGGCGACGTAGCAC (downstream) with an annealing temperature of

55 °C yielded a PCR product of 350 bp.

PCR reactions were run as described (Wordinger et al. 1998; Wordinger et al.

1999; Wordinger et al. 2000; Lambert et al. 2001; Wordinger et al 2002) using Taq Start

Antibody Hot Start with the following cycle conditions: 2 minutes at 94°C, 2 minutes at

92°C, and 40 cycles of 30 seconds at the optimal annealing temperature, extension for 90 seconds at 72°C and denaturation for 45 seconds at 92°C. The amplified PCR products were examined by horizontal electrophoresis in 1.5% agarose gels. To ensure specificity

of the RT-PCR products, Southern blot analysis was performed with probes designed

using Oligo 4.0 that hybridized to a region within the amplified PCR product. PCR

products were sequenced to verify the specificity of the PCR reactions. Table 2 lists the members of the BMP family that are expressed in the human TM and ONH.

Table 1. PCR Primer Pairs, Annealing Temperature and Amplimer Size of BMPs

Table 2. BMP Family Members Expressed in Human TM and ONH

Western immunoblotting: Protein was extracted from cultured cells using lysis

buffer, and proteins were separated by denaturing polyacrylamide gel electrophoresis prior

to electrophoretic transfer to nitrocellulose membranes (Lambert et al. 2001). The membranes were blocked with 5% milk (for BMPs) or 3% gelatin (for BMPRs) and incubated with the following primary antibodies: BMP2, BMP4, BMP5, BMP7 (all from

Santa Cruz, Santa Cruz, CA), or BMP-RIA, BMP-RIB, BMP-RII (from Jackson Immuno

Research, West Grove, PA). The membranes were washed, incubated with secondary

antibodies (goat anti-mouse IgG-horseradish peroxidase for BMPs, Santa Cruz; donkey

anti-goat-horseradish peroxidase for BMP receptors, Jackson immuno Research), and

developed using the WesternBreeze chemiluminescence immunodetection system

(Invitrogen, Carlsbad, CA).

Expression of BMPs, BMPRs mRNA in human TM cells and tissues: Amplification

products of expected for BMP-2, BMP-4, BMP-5 and BMP-7 primer pairs in human TM cells and tissues are shown in FIG. 6. Southern blots using specific probes verified that these were the expected PCR products. All human TM cell lines and tissues expressed message for BMP-2, BMP-4, and BMP-7. However, message for BMP-5 was low to

undetectable in human TM tissue samples (FIG. 6, lanes 6 and 7). Control reactions

without cDNA did not result in amplification products indicating that reagents and primers

were free of DNA or RNA contamination (FIG. 6, lane C).

FIG. 7 shows the amplification products of expected size for BMP-RIA, BMP-RIB,

and BMP-RII primer pairs in human TM cells and tissues. All human TM cells and tissues

expressed message for the BMP receptor complexes. Southern blots using specific probes

verified that these were the expected PCR products. An alternate amplification product

(350 bp) was detected in the BMP-RII PCR reaction. The alternate amplification product was present in all human TM cells and tissues. This alternate band is currently being

identified to determine if it is an alternate spliced form of the receptor. Control reactions without cDNA did not result in amplification products (FIG. 7, lane C) indicating that

reagents and primers were free of DNA or RNA contamination. Expression of BMP and BMP receptor mRNA in human ONH cells and tissues:

Amplification products of expected size for BMP-2, BMP-4, BMP-5 and BMP-7 primer

pairs in human ONH astrocytes and ONH tissues are shown in FIG. 8. All ONH astrocytes

and ONH tissue expressed message for the respective BMP. Human brain astrocytes were

used as a positive control cell line. Southern blots using specific probes verified that these

were the expected PCR products. With the exception of BMP-2, all other BMP were

expressed by human brain astrocytes (FIG. 8, lane 7). Control reactions without cDNA did

not result in amplification products (FIG. 8, lane C) indicating that reagents and primers

were free of DNA or RNA contamination. FIG. 9 shows the amplification products of expected sizes for BMP-2, BMP-4,

BMP-5 and BMP-7 primer pairs in cultured human LC cells. All LC cell lines expressed

message for each BMP. Southern blots using specific probes verified that these were the

expected PCR products. Control reactions without cDNA did not result in amplification

products (FIG. 9, lane C) indicating that reagents and primers were free of DNA or RNA contamination.

Amplification products of expected size for BMP-RIA, BMP-RIB, and BMP-RII

primer pairs in human ONH astrocytes and ONH tissues are shown in FIG. 10. All ONH

astrocyte cell lines and tissues expressed message for BMP-RIA and BMP-RJJB. Southern

blots using specific probes verified that these were the expected PCR products. With the exception of ONH tissue (FIG. 10, lane 6), BMP-RII was expressed by all ONH astrocyte

cell lines. Message for all BMP receptors (FIG. 10, lane 7) was expressed by a human

brain astrocyte cell line that served as a positive control. There appears to be a discrepancy in the expression of BMP-RII in ONH tissue and ONH cell lines. The reduced expression in ONH tissue may reflect a low level of expression. Control reactions without

cDNA did not result in amplification products (FIG. 5, lane C) indicating that reagents and

primers were free of DNA or RNA contamination.

FIG. 11 shows the amplification products of expected size for BMP-RIA, BMP-

RIB, and BMP-RII primer pairs in cultured human LC cells. All LC cell lines expressed

message for each BMP receptor. Southern blots using specific probes verified that these

were the expected PCR products. Control reactions without cDNA did not result in

amplification products (FIG. 11, lane C) indicating that reagents and primers were free of DNA or RNA contamination. Expression of BMP proteins and BMP receptor proteins in human TM and ONH

cells and tissues: FIG. 12 represents chemiluminescent immunoblot detection of BMP-2,

BMP-4, BMP-5, BMP-7, BMP-RIA, BMP-RIB, and BMP-RII proteins in human TM and

ONH cells and tissues. All cell lines studied expressed the respective BMP proteins. The

BMP proteins were detected in cell lines the following molecular weights: 54-56 kDa for

BMP-2, 25-27 kDa for BMP-4, 55-57 kDa for BMP-5, and 77 kDa for BMP-7. Multiple

bands were detected for BMP-2 and BMP-4, which most likely represent glycosylated, and

partially glycosylated forms of these BMPs as seen in other studies. However, we did not

do glycosylation studies as they were beyond the scope of this study. The BMP receptor proteins were detected in cell lines at molecular weights: 38 kDa for BMP-RIA, 64 kDa

for BMP-PJB, and 57 kDa for BMP-RII. Multiple bands were detected for BMP-RIB and BMP-RII in the TM cells, which most likely represent glycosylated, and partially

glycosylated forms as seen in other studies. The expression levels of proteins for the BMP receptors appeared to be lower in the TM cells compared to ONH cells. For example BMP-RII was not detected in TM cells and BMP-RIB was greatly reduced.

Expression of BMP associated protein mRNAs in cultured human TM cells and in

human ONH cells: Amplification products of expected size for BMP associated protein

primer pairs in human TM cell lines are shown in FIG. 13. Human TM cell lines

expressed message for DRM (gremlin), chordin, follistatin, and NMA (BAMBI).

Southern blots using specific probes verified that these were the expected PCR products.

There was no apparent difference in message expression between cell lines. All human

TM cells examined failed to express mRNA for the BMP associated proteins noggin and

Cer-1. Control reactions without cDNA did not result in amplification products indicating

that reagents and primers were free of DNA or RNA contamination. Amplification products of expected size for BMP associated protein primer pairs in

ONH astrocytes and LC cell lines are shown in FIG. 14. All ONH astrocytes and LC cell

lines expressed message for DRM (gremlin), follistatin and NMA (BAMBI). Southern

blots using specific probes verified that these were the expected PCR products. The

majority of LC cells and ONH astrocytes expressed message for chordin. All human ONH

astrocytes and LC cells examined failed to express mRNA for the BMP associated proteins

noggin and Cer-1. Control reactions without cDNA did not result in amplification

products indicating that reagents and primers were free of DNA and RNA contamination.

FIG. 15 shows increased expression of the BMP antagonist gremlin (CKTSFIBI)

in glaucomatous TM cells. Gene expression was assessed using Affymetrix gene arrays (Affymetrix gene chip U133A).

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the

compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied

to the compositions and/or methods and in the steps or in the sequence of steps of the

method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both

chemically and structurally related may be substituted for the agents described herein to

achieve similar results. All such substitutions and modifications apparent to those skilled

in the art are deemed to be within the spirit, scope and concept of the invention as defined

by the appended claims. References

The following references, to the extent that they provide exemplary procedural or

other details supplementary to those set forth herein, are specifically incorporated herein

by reference.

Books

Birren, et al., GENOME ANALYSIS, Vol. 2, (1998).

Clark AF, Browder S, Steely HT, Wilson K, Cantu-Crouch D, McCartney MD, "Cell biology of the human lamina cribrosa," In Drance SM (ed). OPTIC NERVE IN

GLAUCOMA. Kugler Publications, New York: pp. 79-105 (1995b).

Cummings, Michael R., HUMAN HEREDITY, Fourth Edition, (1997).

Grierson I, Calthorpe CM, "Characteristics of meshwork cells and age changes in the

outflow system of the eye: their relevance to primary open angle glaucoma." In

Mills KB (ed). GLAUCOMA. PROCEEDINGS OF THE FOURTH INTERNATIONAL

SYMPOSIUM OF THE NORTHERN EYE INSTITUTE, Manchester, UK, New York,

Pergamon: pp. 12-31 (1988).

Hernandez M, Gong H, "Extracellular matrix of the trabecular meshwork and optic nerve

head." in Ritch R., Shields, M.B., Krupin, T. (eds). THE GLAUCOMAS, 2^nd ed. St

Louis: Mosby-Year; pp. 213-249 (1996).

Jorde, et al, MEDICAL GENEΉCS, Second Edition, (1999).

Lutjen-Drecoll E., Rohen J.W., "Morphology of aqueous outflow pathways in normal and

glaucomatous eyes," in Ritch R., Shields, M.B., Krupin, T. (eds). THE

GLAUCOMAS, 2^nd ed. St Louis: Mosby-Year; pp. 89-123 (1996).

Strachan, et al, HUMAN MOLECULAR GENEΉCS, (1996). Tripathi RC, Borisuth NS, Li, J, Tripathi BJ, "Clinical implications of aqueous humor

growth factors in glaucoma," in Ritch R., Shields, M.B., Krupin, T. (eds). THE

GLAUCOMAS, 2^nd ed. St Louis: Mosby-Year; pp. 71-87 (1996).

Varma R, Minckler D, "Anatomy and pathophysiology of the retina and optic nerve." in

Ritch R., Shields, M.B., Krupin, T. (eds). THE GLAUCOMAS, 2^nd ed. St Louis:

Mosby-Year; pp. 139-175 (1996).

Vaughan, D. et al, In: GENERAL OPHTHALMOLOGY, Appleton & Lange, Norwalk, Conn.,

pp. 213-230 (1992).

Other Publications

Agarwal et al, IOVS 38(4):S563 (1997)

Agarwal R, Talati M, Lambert W, Clark AF, Wilson SE, Agarwal N, Wordinger RJ,

"FAS-activated apoptosis and other apoptosis mediators in human trabecular

meshwork cells," Exp. Eye Res. 68:583-590 (1999).

Astrom, A.K., Jin, D., Imamura, T., Roijer, E., Rosenzweig, B., Miyazono, K., ten Dijke,

P., Stenman, G., Mamm. Genome 10(3):299-302 (1999).

Attisano L, Tuen Lee-Hoeflich S, "The Smads," Genome Biol. 2:REVIEWS3010 (2001).

Bengtsson, B., Br. J. Ophthalmol 73:483-487 (1989).

Chang B, Smith RS, Peters M, Savinova DV, Hawes NL, Zabalata A, Nusinowitz S,

Martin JE, Davisson ML, Sepko CL, Hogan BML, John SWM, "Haploinsufficient

Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated

intraocular pressure," BMC Genetics 2:18 (2001).

Chundru RK, Agarwal R, Wordinger RJ, Whitson JT, "Detection of neurotrophins in

human aqueous humor," Invest. Ophthalmol. Vis. Sci. 4 S236 (2000). Clark AF, Kawase K, English- Wright S, Lane D, Steely HT, Yamamoto T, Kitazawa Y,

Kwon YH, Fingert JH, Swiderski RE, Mullins RF, Hageman GS, Alward WLM,

Sheffield VC, Stone EM, "Expression of the glaucoma gene myocilin (MYOC) in

the human optic nerve head " FASEB J. 15:1251-1253 (2001).

Clark AF, Lane D, Wilson K, Miggans ST, McCartney MD, "Inhibition of dexamethasone-

induced cytoskeletal changes in cultured human trabecular meshwork cells by

tetrahydrocortisol " Invest. Ophthalmol. Vis. Sci. 35:805-813 (1996).

Clark AF, Miggans ST, Wilson K, Browder S, McCartney MD, "Cytoskeletal changes in

cultured human glaucoma trabecular meshwork cells," J. Glaucoma 4:183-188 (1995c).

Clark AF, Steely HT, Dickerson JE, English-Wright S, Stropki K, McCartney MD, Jacobson N, Shepard AR, Clark JI, Matsushima H, Peskind ER, Leverenz JB,

Wilkinson CW, Swiderski RE, Fingert JH, Sheffield VC, Stone EM, "Glucocorticoid induction of the glaucoma gene MYOC in human and monkey

trabecular meshwork cells and tissues," Invest. Ophthalmol. Vis. Sci. 42:1769-

1780 (2001b).

Clark AF, Wilson K, de Kater AW, Allingham RR, McCartney MD, "Dexamethasone-

induced ocular hypertension in perfusion-cultured human eyes," Invest.

Ophthalmol. Vis. Sci. 36:478-489 (1995a).

Clark AF, Wilson K, McCartney MD, Miggans ST, Kunkle M, Howe W, "Glucocorticoid-

induced formation of cross-linked actin networks in cultured human trabecular

meshwork cells," Invest. Ophthalmol. Vis. Sci. 35:281-294 (1994). Dickerson JE, Steely HT, English- Wright SL, Clark AF, "The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells,"

Exp. Eye Res. 66:731-738 (1998).

Dudley AT, Lyons KM, Robertson EJ, "A requirement for bone morphogenic protein-7

during development of the mammalian kidney and eye," Genes Dev. 9:2795-2807

(1995). Furuta Y, Hogan BL, "BMP4 is essential for lens induction in the mouse embryo," Genes

Dev. 12:3764-3775 (1998). Greve, M. et al, Can. J. Ophthamol 28:201-206 (1993). Giguere et al, Cell 46:645-652 (1986).

Hernandez MR, Andrzejewska WM, Neufeld AH, "Changes in the extracellular matrix of

the human optic nerve head in primary open-angle glaucoma," Am. J. Ophthalmol.

109:180-188 (1990).

Hernandez MR, Pena JD, "The optic nerve head in glaucomatous optic neuropathy," Arch

Ophthalmol. 115:389-395 (1997).

Hitchings, R. A., Br. J. Ophthamol. 77:326 (1993).

Hogan BL, "Bone morphogenic proteins: multifunctional regulators of vertebrate

development," Genes Dev. 10:1580-1594 (1996).

Hu DN, Ritch R, "Hepatocyte growth factor is increased in the aqueous humor of

glaucomatous eyes," J. Glaucoma 10:152-157 (2001).

Inatani M, Tanihara H, Katsuta H, Honjo M, Kido N, Honda Y, "Transforming growth

factor beta 2 levels in aqueous humor of glaucomatous eyes," Graefes Arch. Clin.

Exp. Ophthalmol. 239:109-113 (2001).

Itoh et al, Eur. J. Biochem. 267:6954-6967 (2000). Jena N, Martin-Seisdedos C, McCue P, Croce CM, "BMP7 null mutation in mice:

developmental defects in skeleton, kidney, and eye," Exp. Cell Res. 230:28-37

(1997).

Kawabata et al, Cytokine & Growth Factor Review, 9:49-61 (1998).

Kerrigan LA, Zack DJ, Quigley HA, Smith SD, Pease ME, "TUNEL-positive ganglion

cells in human primary open-angle glaucoma," Arch. Ophthalmol. 115:1031-1035

(1997).

Lambert et al, IOVS 38(4):S162 (1997).

Lambert W, Agarwal R, Howe W, Clark AF, Wordinger RJ, "Neurotrophin and

neurotrophin receptor expression by cells of the human lamina cribrosa " Invest.

Ophthalmol. Vis. Sci., 42:2315-2323 (2001).

Leske, M. C. et al, Amer. J. Epidemiol. 113:1843-1846 (1986).

Liu et al, 7O S 40(4):S673 (1999).

Liu Y, Belayev L, Zhao W, Busto R, Saul I, Alonso O, Ginsberg MD, "The effect of bone

morphogenic protein-7 (BMP-7) on functional recovery, local cerebral glucose

utilization and blood flow after transient focal cerebral ischemia in rats," Brain

Res. 905:81-90 (2001).

Liu X, Lambert W, Agarwal R, Talati M, Cross W, Clark AF, Wordinger RJ, "Human

trabecular meshwork cells express the ciliary neurotrophic factor (CNTF)

tripartate receptor complex " Exp. Eye Res. 72:711-717 (2001).

Luo G, Gofmann C, Bronckers AL, Sohocki M, Bradley A, Karsenty G, "BMP-7 is an

inducer of nephrogenesis, and is also required for eye development and skeletal

patterning," Genes Dev. 9:2808-2820 (1995). McMahon, R., Murphy, M., Clarkson, M., Taal, M., Mackenzie, H.S., Godson, C, Martin,

F., Brady, H.R., J. Biol. Chem. 275(14):9901-9904 (2000).

Miyazono. J. Cell Science, 113:1101-1109 (2000).

Mohan RR, Kim WJ, Mohan RR, Chen L, Wilson SE, "Bone morphogenic proteins 2 and

4 and their receptors in the adults human cornea," Invest. Ophthalmol. Vis. Sci.

39:2626-2636 (1998).

Morrison JC, Dorman-Pease ME, Dunkelberger GR, Quigley HA, "Optic nerve head

extracellular matrix in primary optic atrophy and experimental glaucoma," Arch.

Ophthalmol. 108:1020-1024 (1990). Murphy, M., Godson, C, Cannon, S., Kato, S., Mackenzie, H.S., Martin, F., Brady, H.R.,

J. Biol. Chem. 274(9):5830-5834 (1999). Nickel j, Dreyer MK, Kirsch T, Sebold W, "The crystal structure of BMP-2.BMPR-1A

complex and the generation of BMP-2 antagonists," J. Bone & Joint Surgery 83

A(suppl 1):S1-S7 (2001).

Nohno, T., Ishikawa, T., Saito, T., Hosokawa, K., Noji, S., Wolsing, D.H., Rosenbaum,

J.S., J. Biol. Chem. 270(38):22522-22526 (1995). Nonner D, Barrett EF, Kaplan P, Barrett JN, "Bone morphogenic proteins (BMP6 and

BMP7) enhance the protective effect of neurotrophins on cultured septal

cholinergic neurons during hypoglycemia," J. Neurochem. 77:691-699 (2001).

Obata H, Kaji Y, Yamada H, Kato M, Tsuru T, Yamashita H, "Expression of tranfsorming

growth factor-beta superfamily receptors in rat eyes," Acta. Ophthalmol. Scand.

77:151-156 (1999).

Pang I-H, McCartney MD, Steely HT, Clark AF, "Human ocular perfusion organ culture:

a versatile ex vivo model for glaucoma research," J. Glaucoma 9:468-479 (2000). Pena JD, Taylor AW, Ricard CS, Vidal I, Hernandez MR, "Transforming growth factor

beta isoforms in human optic nerve heads," Br. J. Ophthalmol. 83:209-218 (1999).

Picht G, Welge-Luessen U, Grehn F, Lutjen-Drecoll E, "Transforming growth factor beta

2 levels in the aqueous humor in different types of glaucoma and the relation to

filtering bleb development," Graefes Arch. Clin. Exp. Ophthalmol. 239:199-207

(2001).

Quigley HA, McKinnon SJ, Zack DJ, Pease ME< Kerrigan-Baumrind LA, Kerrigan DF,

Mitchell RS, "Retrograde axonal transport of BDNF in retinal ganglion cells is

blocked by acute IOP elevation in rats," Invest. Ophthalmol. Vis. Sci. 41 :3460-

3466 (2000).

Quigley HA, "Neuronal death in glaucoma," Prog. Retin. Eye Res. 18:39-57 (1999).

Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, Zack DJ, "Retinal

ganglion cell death in experimental glaucoma and after axotomy occurs by

apoptosis," Invest. Ophthalmol. Vis. Sci. 36:774-786 (1995).

Reddi AH, "Bone morphonegetic proteins: an unconventional approach to isolation of

first mammalian morphogens," Cytokine Growth Factor Rev. 8:11-20 (1997).

Reddi AH, "Bone morphogenic proteins and skeletal development: the kidney-bone

connection," Pediatr. Nephrol. 14:598-601 (2000).

Rohen JW, "Why is intraocular pressure elevated in chronic simple glaucoma?

Anatomical considerations." Ophthalmology 90:758-765 (1983).

Steely HT, Browder SL, Julian MB, Miggans ST, Wilson KL, Clark AF, "The effects of

dexamethasone on fibronectin expression in cultured human trabecular meshwork

cells ," Invest. Ophthalmol. Vis. Sci. 33: 2242-2250 (1992). Steely HT, English- Wright SL, Clark AF, "Similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma," Exp. Eye Res. 70:17-

30 (2000). Strong, N. P., Ophthal. Physiol. Opt. 12:3-7 (1992).

ten Dijke, P.P., Ichijo, H, Franzen, P., Schulz, P., Saras, J., Toyoshima, H., Heldin, C.H.,

Miyazono, K., Oncogene 8(10):2879-2887 (1993).

Tripathi RC, Borisuth NS, Kolli SP, Tripathi BJ, "Trabecular cells express receptors that

bind TGF-beta 1 and TGF-beta 2: a qualitative and quantitative characterization,"

Invest. Ophthalmol. Vis. Sci. 34:260-263 (1993b).

Tripathi RC, Borisuth NS, Tripathi BJ, "Detection, quantification, and significance of basic fibroblast growth factor in the aqueous humor of man, cat, dog and pig,"

Exp. Eye Res. 54:447-454 (1992). Tripathi RC, Borisuth NS, Tripathi BJ, Fang VS, "Analysis of human aqueous humor for epidermal growth factor," Exp. Eye Res. 53:407-409 (1991).

Tripathi RC, Chan WF, Li J, Tripathi BJ, "Trabecular cells express the TFG-beta 2 gene

and secrete the cytokine," Exp. Eye Res. 58:523-528 (1994a).

Tripathi RC, Li J, Borisuth NS, Tripathi BJ, "Trabecular cells of the eye express messenger RNA for transforming growth factor-beta 1 and secrete this cytokine,"

Invest. Ophthalmol. Vis. Sci. 34:2562-2569 (1993a).

Tripathi RC, Li J, Chan WF, Tripathi BJ, "Aqueous humor in glaucomatous eyes contains

an increased level of TFG-beta 2," Exp. Eye Res. 59:723-727 (1994c).

Tripathi RC, Li J, Tripathi BJ, "Immunolocalization ofbFGF in the trabecular meshwork

and detection of its mRNA in trabecular cells," Exp. Eye Res. 58:503-507 (1994b). Trousse F, Esteve P, Bovolenta P, "BMP4 mediates apoptotic cell death in the developing

chick eye," J. Neurosci. 21 : 1292-1301 (2001).

Tuck, M. W. et al, Ophthal. Physiol. Opt. 13:227-232 (1993).

Vernon, S. A., Eye 7: 134-137 (1993).

Von Bubnoff A, Cho KW, "Intracellular BMP signaling regulation in vertebrates:

pathway or network?" Dev. Biol. 239:1-14 (2001).

Wang W-H, McNatt LG, Shepard AR, Jacobson N, Nishimura DY, Stone ΕM, Sheffield

VC, Clark AF, "Optimal procedure for extracting RNA from human ocular tissues

and expression profiling of the congenital glaucoma gene FOXC1 using

quantitative RT-PCR," Molecular Vision 7:89-94 (2001).

Wilson K, McCartney MD, Miggans ST, Clark AF, "Dexamethasone induced

ultrastructural changes in cultured human trabecular meshwork cells," Current

Eye Research 12:783-793 (1993).

Wordinger et al, IOVS 40(4):S504 (1999a).

Wordinger RJ, Agarwal R, Talati M, Fuller J, Lambert W, Calrk AF, "Expression of bone

morphogenic proteins (BMP), BMP receptors, and BMP associated proteins in

human trabecular meshwork and optic nerve head cells and tissues," Molec. Vision

8:241-256 (2002).

Wordinger RJ, Clark AF, Agarwal R, Lambert W, McNatt L, Wilson SΕ, Qu Ε, Fung BK-

K, "Cultured human trabecular meshwork cells express functional growth factor

receptors " Invest. Ophthalmol. Vis. Sci. 39: 1575-1589 (1998).

Wordinger RJ, Clark AF, Agarwal R, Lambert W, Wilson SΕ, "Expression of alternatively

spliced growth factor receptor isoforms in the human trabecular meshwork,"

Invest. Ophthalmol. Vis. Sci. 40:242-247 (1999b). Wordinger RJ, Lambert W, Agarwal R, Talati M, Clark AF, "Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk)," Invest.

Ophthalmol. Vis. Sci. 41:3833-3841 (2000).

Yamashita H, Ten Dijke P, Heldin CH, Miyazono K, "Bone morphogenic protein

receptors," Bone 19:569-574 (1996).

You L, Kruse FE, Pohl J, Volcker HE, "Bone morphogenic proteins and growth and differentiation factors in the human cornea," Invest. Ophthalmol. Vis. Sci. 40:296-

311 (1999). Zhang D, Mehler MF, Song Q, Kessler JA, "Development of bone morphogenic protein

receptors in the nervous system and possible roles in regulating trkC expression,"

J. Neurosci. 18:3314-3326 (1998).

Claims (7)

We Claim:

1. A method for diagnosing glaucoma in a sample obtained from a cell or bodily fluid

by detecting altered expression of a bone morphogenic family member gene, said

method comprising the steps of:

g) obtaining a tissue or fluid sample from a patient suspected of having

glaucoma;

h) extracting DNA from said sample;

i) obtaining a plurality of PCR primers, wherein said primers each comprise a

sequence consisting of from 18 to 1547 contiguous nucleotides from SEQ

ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:37,

SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID

NO:47, or SEQ ID NO:53;

j) amplifying regions of the extracted DNA using said primers to obtain a

PCR product;

k) resolving the PCR product; and

1) identifying differences between the sequence of the amplified extracted

DNA and the sequence of the primer;

where a difference between the amplified sequence and the primer is diagnostic of

glaucoma.

2. The method of claim 1, wherein said tissue or fluid sample is blood or buccal cells.

3. The method of claim 1, wherein the primers comprise sequences consisting of from

20 to 100 contiguous nucleotides of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID

NO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,

SEQ ID NO:47, or SEQ ID NO:53.

4. The method of claim 1, wherein the primers comprise sequences consisting of from

20 to 50 contiguous nucleotides of SEQ ID NO:3.

5. The method of claim 1, wherein the PCR product is resolved by SSCP, DGGE,

ASO or RFLP.

6. A method for treating glaucoma comprising administering to a patient in need

thereof a composition comprising a sequence consisting of at least one compound

selected from the group consisting of a BMP2 agonist, a BMP4 agonist, a BMP5

agonist, a BMP7 agonist, a Smad 1/5 agonist, a chordin antagonist, a gremlin

antagonist and a follistatin antagonist.

7. A method for identifying a therapeutic agent for the treatment of glaucoma, said

method comprising: d) obtaining a first composition comprising a population of recombinant cells

expressing BMP-2A, BMP4, BMP-5, or BMP7; e) obtaining a candidate substance;

f) incubating said composition and said candidate substance; g) testing said composition for its ability to turn on BMP-induced Smad

signaling pathways or BMP-regulated gene expression; and

h) identifying a candidate substance that inhibits, or stimulates, BMP-induced

Smad signaling pathways or BMP-regulated gene expression.