Lens epithelial cell derived growth factor

Details Lens epithelial cell derived growth factor Lens epithelial cell derived growth factor

1998-07-23

2004-06-15

Romeo, David S. (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S325000, C435S810000, C536S023500, C536S024100

Reexamination Certificate

active

06750052

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acids and encoded polypeptides of a lens epithelial cell derived growth factor (LEDGF), and diagnostics and therapeutics related to medical conditions associated with such genes and polypeptides, including cataracts.
BACKGROUND OF THE INVENTION
The term cataract is used to define the opacification of the normally clear and transparent crystalline lens. Several types of cataract etiology have been described. For example, congenital cataracts occur as a complication of intrauterine rubella, herpes simplex, herpes zoster, syphilis and cytomegalic inclusion disease, the majority being idiopathic or inherited. Acquired cataracts result from trauma, radiation, drugs, metabolic disorders, ocular inflammatory disorders, or aging (senile or age-related cataracts). It has been estimated that nearly one billion elderly people throughout the world have age-related cataracts [ARCs] (Thylefors et al.,
Available Data on Blindness
, Geneva, Switzerland; WHO Program for the Prevention of Blindness, 1994). The underlying mechanisms responsible for ARCs, however, are not well understood.
Although the anterior lens is covered with a single layer of cuboidal lens epithelial cells (LECs), most of the remaining lens is composed of anucleated fiber cells. With age, even though the lens enlarges, the epithelial cell density and cytological activity decline (Guggenmoos-Holzmann et al.,
Invest. Ophthalmol. Vis. Sci
. 30:330-332, 1989, Konofsky et al.,
Ophthalmology
94:875-880, 1987, and Straatsma et al.,
Am. J. Ophthalmol
. 112:283-296 1991); this decrease is most pronounced in lenses with cortical and mixed cataracts. Also in these cataracts, there is greater LEC metaplasia than in the normal lens (Saitoh et al.,
Nippon Ganka Gakkai Zasshi
94:176-180, 1990, Streeten and Eshaghian,
Arch. Ophthalmol
. 96:1653-1658, 1978, and von Sallmann,
Am. J. Ophthalmol
. 44:159-170, 1957). Since the LECs maintain lens homeostasis, a decrease in epithelial cell number or metabolic activity is expected to disturb normal lens physiology.
For survival, the cells require growth or proliferation factors, and several such factors have been reported: neurotrophic factor for neurons (Cowan et al.,
Science
225:1258-1264, 1984, Purves,
Body and brain. A trophic theory of natural connections
. Harvard University Press. Cambridge, Mass. pp. 231, 1988, Barde,
Neuron
2:1525-1534, 1989, and Oppenheim,
Science
240:919-922, 1991), colony stimulation factor for myeloid cells (Metcalf,
Nature
339:27-30, 1989, Williams et al.,
Nature
343:76-78, 1990, and Koury and Bondurant,
Science
248:378-381, 1990), specific hormones for endocrine-dependent cells (Kerr and Searle,
Virchows. Arch. B. Cell Pathol
. 13:87-92, 1973, Krypaniou and Issacs,
Endocrinology
122:552-562, 1988, and Wyllie et al.,
J. Pathol
. 111:85-94, 1973), and specific growth factors for oligodendrocytes (Barres et al.,
Cell
70:31-46, 1992). Ishizaki et al. predicted that LECs must have such growth factors (
J. Cell Biol
. 121:899-908, 1993].
It was demonstrated recently (Ibaraki et al.,
Exp. Eye Res
. 64:229-238, 1997 and Singh et al.,
J. Immunol
. 155:993-999, 1995), that auto-antibodies against lens P-crystallins induced LEC damage and cataract formation in mice. Both serum and monoclonal antibody (Ab) transfer studies established that humoral rather than cellular immunity was responsible for the death of LECs. It was found that anti-P crystallin antibodies (Abs) killed LECs and that damage was age-dependent (Singh et al.,
J. Immunol
. 155:993-999, 1995). In human serum, anti-lens Abs were more prevalent in patients with age-related cataracts (98%) than in subjects with clear lenses (40%) (Singh et al.,
Exp. Eye Res
. Submitted, 1997). It was also shown that more than 96% of the sera from patients with ARC, but fewer than 30% with clear lenses, were cytotoxic. Mixing cytotoxic anti-lens Abs with whole lens antigens (Ags) decreased or eliminated the cytotoxic effects. These findings raise the possibility that human ARCs are caused by an autoimmune insult to the LECs.
There exists a need to influence favorably the physical properties of the crystalline lens.
There also exists a need to identify the gene(s) responsible for cataract and age-related cataract in particular, and to provide therapy for preventing and treating cataracts.
An object of the invention is to provide compounds that desirably influence the physical properties of the crystalline lens.
Another object of the invention is to provide therapeutics for treating diseases or conditions involving LEDGF expression.
Still another object of the invention is to provide diagnostics and research tools relating to LEDGF. These and other objects will be described in greater detail below.
SUMMARY OF THE INVENTION
We describe herein the molecular cloning and characterization of LEDGF, a novel polypeptide that stimulates protein synthesis. This increase in protein synthesis leads to the enhanced growth of a variety of cell types such as those of epithelial, epidermal or kidney origin, including lens epithelial cells. Anti-LEDGF Abs blocked the stimulatory effects of this protein, resulting in the death of LECs and other types of cells, including neuronal, fibroblasts, etc. Increased levels of anti-LEDGF Abs were found in patients with ARCs, suggesting that neutralization of LEDGF by auto-Abs is responsible for the development of cataracts and ARCs in particular.
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and agents which bind such polypeptides, including antibodies. The foregoing can be used, inter alia, in the diagnosis or treatment of conditions characterized by the aberrant expression of a LEDGF nucleic acid or polypeptide. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, we present the cDNA cloning of a 61 kDa protein, LEDGF, which stimulates proliferation of a number of different cell types, particularly those of epithelial character, and include lens epithelial cells.
According to one aspect of the invention, isolated nucleic acid molecules that code for a lens epithelial cell derived growth factor polypeptide are provided and include: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid of SEQ ID NO:1 and which code for a lens epithelial cell derived growth factor polypeptide, (b) deletions, additions and substitutions of (a) which code for a respective lens epithelial cell derived growth factor polypeptide, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule comprises nucleotides 1-3360 of SEQ ID NO:1. In some embodiments the isolated nucleic acid molecules are those comprising the human cDNA or gene corresponding to SEQ ID NO:13. The isolated nucleic acid molecule also can comprise a molecule which encodes the polypeptide of SEQ ID NO:2.
The invention in another aspect is an isolated nucleic acid molecule selected from the group consisting of (a) a fragment of a nucleic acid molecule of SEQ ID NO:1, of sufficient length to represent a sequence unique within the human genome, and identifying a nucleic acid encoding a Lens Epithelial Cell Derived Growth Factor polypeptide, (b) complements of (a), provided that the fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from the sequence group consisting of (1) sequences having the GenBank accession numbers of Table III, (2) complements of (1), and (3) fragments of (1) and (2).
In one embodiment the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two conti

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