Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-24
2001-10-23
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S325000, C536S023100, C536S024300
Reexamination Certificate
active
06306586
ABSTRACT:
BACKGROUND OF THE INVENTION
A cataract is any clouding or haziness in the eye's natural lens. Most cataracts develop as part of the normal aging process, from a change in the internal composition of the natural lens. They are extremely common, since anyone living beyond 60 or 70 years will most certainly develop cataracts. About 70% of persons over the age of 75 have visually significant cataracts. Cataracts resulting from the aging process usually develop in both eyes, but often at different rates. While most kinds of cataracts progress slowly over several years, a cataract may progress rapidly over several months producing a significant decrease in vision in the affected eye.
Certain types of cataracts are developmental or congenital and are present at birth. These can be due to chromosomal abnormalities. Furthermore, certain diseases can be associated with the development of cataracts. For example, the autosomal dominant disease termed Anterior Segment Mesenchymal Dystrophy (ASMD), is a disease associated with an anterior segment abnormality in which each individual affected develops cataracts, even at an early age (Hittner et al. (1982)
Am. J. Ophthal.
93:57). This disease is also associated with strong defects in visual acuity, corneal opacities, lens opacities, optic nerve abnormalities, and the development of glaucoma.
At present, the only way to restore visual loss from cataracts is surgical removal of the cloudy lens. Although most patients benefit from cataract surgery with improved eyesight, this in not true for everyone. Some people do not have sharp eyesight after the surgery due to other eye diseases such as glaucoma and macular degeneration. As with all surgical operations, sometimes complications can occur resulting in reduced vision post-operatively or even rarely blindness. Thus, it is highly desirable to develop methods for preventing cataracts and methods for treating cataracts which are less invasive.
The development of the lens is a well-studied process and includes several stages. The formation of the lens placode (by 10-dpc in mouse embryo) is induced by the neuroepithelium of the optic vesicle after establishment of close contact between the optic vesicle and overlying surface ectoderm at day 9.5-9.75 pc (Zwaan, J. (1975)
Dev. Biol.
44(2): 306-312; Kaufman, M. H. (1992)
The atlas of mouse development.
Academic Press, London). The lens placode starts to invaginate in the 10.5-dpc embryo and the lens cup rapidly deepens within the next few hours. The closure of the lens cup and detachment of the lens vesicle from the surface occur by day 11-11.25 pc. At day 11.5 pc the formation of lens fibers begins, with continued elongation of the fibers leading to occlusion of the lens cavity before the end of day 13 pc. By day 13 the lens has a similar configuration to that of the adult organ (Zwaan, J. (1975) supra; Kaufman, M. H. (1 992) supra).
During, lens development the peripheral epithelial cells proliferate anterior to the lens equator (or bow region), where they subsequently differentiate into the lens fiber cells. Differentiation into lens fiber cells includes cell elongation and loss of subcellular organelles, cessation of DNA replication and of cell division and synthesis of fiber cell-specific proteins such as various crystallins (Reneker, L. W. and Overbeek, P. A. (1996)
Dev. Biol.
180(2): 554-565).
Many genes are involved in lens formation. Pax-6, a master gene in eye development, has been implicated in the various mouse and rat Small eye (Sey) mutant phenotypes (Hill, R. E., et al. (1991)
Nature
354(6354):522-525) as well as in human aniridia (Jordan, T., et al. (1992)
Nature Genet.
1(5):328-332). Pax6 is expressed in all stages of lens development. A histologic analysis of the murine homozygous Sey mutants revealed that the optic vesicles grow out but there is no lens induction (Hogan, B. L., et al. (1988)
Development
103 Suppl. 115-119). Tissue transplantation experiments in a rat Sey mutant demonstrated that homozygous rSey ectoderm loses its lens-forming competence early in development (Graw, J. (1996)
Dev. Genet.
18(3):181-197). Several papers have demonstrated that Pax6 is involved in the regulation of lens-specific expression of the crystallin genes (Cvekl, A. and Piatigorsky, J. (1996)
Bioessays
18(8):621-630). These results suggest that Pax-6 is involved in lens induction and subsequent development and differentiation.
Other homeobox genes involved in early lens development include Prox1 (related to Drosophila prospero), which is expressed in the early lens placode and later throughout the lens, especially in the bow region, in chicken (Zinovieva, R. D., et al, (l996)
Genomics
35(3): 517-522), and in the developing lens in mouse (Oliver, G., et al. (1993)
Mech. Dev.
14(1):3-16) and human (Zinovieva, R. D., et al. (1996) supra). Murine Six3 is expressed in the optic vesicle and lens (Oliver, G., et al, (1995)
Development
121(12):4045-4055). In Xenopus, the Six3 transcript was detected in the anterior neural plate, a region involved in lens induction. The ectopic expression of murine Six3 in fish embryos resulted in ectopic lens formation in the area of the otic vesicle (Oliver, G., et al, (1996)
Mech. Dev.
60(2):233-239). Chicken GH6 is expressed in the lens epithelium at stage 23, which is roughly equivalent to mouse day 11 pc (Stadler, H. S. and Solursh, M. (1994)
Dev. Biol.
151(1):251-262). Mouse Msx2 and Emx1 are expressed in the lens, as reviewed by Beebe (Beebe, D. C. (1994)
Invest. Ophthalmal. Vix. Sci.
35:2897-2900). In addition, some Sox homeodomain proteins have been shown to be involved in lens-specific activation of crystallin genes in mouse (Kamachi, Y., et al, (1995)
EMBO J.
15:3510-3519).
However, none of these genes have been involved in the development of cataracts. It would be highly desirable to isolate genes which are associated with ocular diseases or disorders, such as cataracts and which would thus provide diagnostic and therapeutic methods. In particular, therapeutic methods less intrusive than surgery could be developed.
SUMMARY OF THE INVENTION
The invention is based at least in part on the isolation of DNA molecules encoding Pitx3 polypeptides, such as human and mouse Pitx3 polypeptides. As described herein, Pitx3 proteins are homeobox containing proteins, which are involved in the development of the lens and contribute to diseases and disorders of the lens, such as cataracts. In fact, as shown herein, a mutated form of Pitx3 having a different C-terminal sequence from that of the wild-type Pitx3 protein (shown in
FIGS. 4 and 5
) was found in all affected individuals of a family carrying the disorder Anterior Segment Mesenchymal Dysgenesis (ASMD), all of which have cataracts. Furthermore, another mutation, i.e., an amino acid substitution in the N-terminus of the protein (shown in
FIG. 6
) was found in an individual having congenital cataract, but not in control individuals. In addition, Pitx3 is expressed predominantly in the lens during embryonic development and the mouse Pitx3 gene is located in the chromosomal region as the mutant aphakia gene, which is associated with a phenotype of small eyes and overall ocular disorganization.
Accordingly, in one aspect, the invention provides Pitx3 nucleic acids. In one embodiment, the Pitx3 nucleic acid comprises a nucleotide sequence set forth in SEQ ID Nos. 1, 2, 4, 5, 6, 8 and/or 29 or complement thereof In further embodiments, the nucleic acid molecule is a Pitx3 nucleic acid having an overall homology of at least about 70%, 80%, 85%, 90%, 95%, 98% or preferably 99% with a nucleotide sequence shown as SEQ ID NOs. 1, 2, 4, 5, 6, 8 and/or 29 or to the complement of the nucleic acid shown as SEQ ID NOS 1, 2, 4, 5, 6, 8 and/or 29. Also within the scope of the invention are nucleic acids comprising a nucleotide sequence which is homologous, i.e., has a certain degree of homology or identity, with a portion of a nucleotide sequence shown as SEQ ID NOS. 1, 2, 4, 5, 6, 8 and/or 29 or to the complement thereof. The portion can be the nucleo
Murray Jeffrey C.
Semina Elena
Arnold Beth E.
Clauss Isabelle M.
Foley Hoag & Eliot LLP
Jones W. Gary
Souaya Jehanne
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