Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1995-08-29
2002-09-17
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600
Reexamination Certificate
active
06451763
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the purification and use of a retinal pigmented epithelium derived neurotrophic factor (PEDF). This invention also relates to a truncated version of PEDF that is referred to as PEDF-BH. In addition to PEDF and PEDF-BH and functionally equivalent proteins, this invention relates to nucleic acids that encode PEDF, PEDF-BH and functionally equivalent proteins, to vectors comprising such nucleic acids, to host cells into which such vectors have been introduced, and to the use of these host cells to produce such proteins.
BACKGROUND OF THE INVENTION
Many types of neurons depend upon the availability of special regulatory molecules, known as neurotrophic factors, for their survival and well-being. The best characterized of the neurotrophic factors is nerve growth factor (NGF). NGF regulates the survival and specialized function of sympathetic and dorsal root ganglion neurons in the peripheral nervous system and of some cholinergic neurons in the central nervous system. Trophic factors, which act on other neurons, have also been identified, and two such factors, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) have been purified. Moreover, it has recently been shown that some growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), which initially were identified based on their mitogenic effects upon cells, also function as survival-promoting agents for some neurons. Post-synaptic target cells and satellite cells, such as glial cells, appear to be major sources of neurotrophic factors.
It has been proposed that the survival of retinal photoreceptor cells may also be regulated by specific neurotrophic factors. Evidence supporting this concept includes the observation that photoreceptors undergo developmental neuronal death in some species, a phenomenon which is generally considered to reflect the limited availability of neurotrophic factors. Photoreceptor development, as well as maintenance of normal function, has also been shown to require interactions with the retinal pigment epithelium (RPE), suggesting that RPE-derived molecules or factors could be necessary for photoreceptor function and survival.
The RPE develops in advance of and lies adjacent to the neural retina. A closed compartment between the two cell layers contains the interphotoreceptor matrix, and many soluble secretory products of RPE and neural retina cells are contained in the interphotoreceptor matrix. Nutrients, metabolites or trophic factors exchanged between the RPE and neural retina, must pass through the interphotoreceptor matrix. RPE cells, for example, are thought to synthesize and secrete a photoreceptor survival-promoting factor (PSPA).
Cultured RPE cells synthesize a number of well known trophic factors, including platelet derived growth factor (PDGF), FGF, transforming growth factor-&agr; (TGF-&agr;), and transforming growth factor-&bgr; (TGF-&bgr;). It is possible that these or other unknown factors derived from RPE could influence the development of the neural retina.
The neural-derived RPE forms a monolayer of cells interposed between the neural retina and circulating blood within the choroid. In this strategic location, the RPE forms a part of the blood-retina barrier, performs functions essential to retinal integrity and functions, and plays important roles in vascular, inflammatory, degenerative, and dystrophic diseases of the retina and choroid. The functions of the RPE in relation to the visual process are several-fold and include light-dark adaption, phagocytosis of shed photoreceptor outer segment membrane and nutrition. On the other hand, the close interdependence of the RPE and the neural retina during normal development has been known for a long time, but functionally is not well understood, although it is known that the RPE is important for retinal regeneration. It has been consistently observed that loss of contact of the neural retina with the RPE of many vertebrates (retinal detachment) results in degeneration of the retina. As a side effect of the retinal detachment, strong cell proliferation, originating from the RPE which underlies the areas of detachment, has often been observed.
Thus, identification of hypothetical survival-promoting factors for photoreceptor cells would potentially be of great importance for the treatment of pathological conditions which result in blindness due to photoreceptor degeneration of unknown etiology. While these types of selective photoreceptor degenerations could be due to a variety of different mechanisms, analogies with neuronal degenerations in other regions of the nervous system suggest the possible involvement of a neurotrophic activity in the retina.
SUMMARY OF THE INVENTION
The present invention relates to a purified retinal pigmented epithelium derived neurotrophic factor composition and a method for purifying such a retinal pigmented epithelium neurotrophic factor. The purification procedure comprises providing an impure protein fraction containing retinal pigmented epithelium derived neurotrophic factor and applying the impure protein fraction containing retinal pigmented epithelium derived neurotrophic factor to a cation-exchange chromatography medium. The cation-exchange chromatography medium is then washed to elute any unbound proteins and the retinal pigmented epithelium derived neurotrophic factor is eluted from the cation-exchange chromatography medium and collected.
The present invention also relates to a recombinant DNA molecule comprising a gene encoding a retinal pigmented epithelium derived neurotrophic factor having the DNA sequence or the amino acid sequence in SEQ ID NO:1 and to an organism transformed with a recombinant DNA molecule comprising a retinal pigmented epithelium derived neurotrophic factor gene having a DNA sequence identified in SEQ ID NO:1.
The present invention also relates to a method of treating tumors, ocular diseases and conditions resulting from the activity of serine proteases which comprises administering PEDF.
DETAILED DESCRIPTION
Cells of the retinal pigmented epithelium are closely associated with differentiating retinoblasts in vivo and contribute to an environment essential to their development and normal function, both in vivo and in vitro. Retinoblastoma cells exhibit a multi-potential differentiative nature paralleling that of their precursor, the primitive retinoblast, as evidenced by the fact that agents, such as laminin, sodium butyrate and dibutyryl cAMP, can induce the expression of neuronal, glial, and pigmented epithelial characteristics in vitro.
A human retinoblastoma cultured cell line, Y79 cells, when exposed to RPE-conditioned medium (RPE-CM), exhibits a high degree of neuronal-like differentiation. The differentiation is observed in both the morphological and biochemical characteristics of the cells. The exposed cells extend arborizing neuritic processes from their cell bodies and express elevated levels of neuron-specific enolase (NSE) and neurofilament proteins. In addition, RPE-CM extends the life of the attached, differentiated cells for more than 30 days as compared to 10 to 15 days for non-treated cells.
RPE-secreted proteins have been fractionated from RPE-CM, and a protein doublet, with an apparent molecular weight of about 50,000 to about 55,000, that is unique to RPE-CM has been identified. This pigment epithelium derived neurotrophic factor (PEDF), which is a major secretory product of human fetal RPE cells, has been isolated and shown to have neurotrophic effects on Y79 retinoblastoma cells. Additionally, a smaller 36,000 molecular weight, form of PEDF has also been identified.
PEDF has uses in the treatment of retinal diseases including, but not limited to, retinoblastoma and other ocular tumors, retinitis pigmentosa, various forms of retinal detachment, macular degeneration, diabetic retinopathy, and other inherited and age-related pathologies of retinal cells.
In the case of retinal tumors, PEDF induces the tumor cells to display biochemical and phenotypic cha
Becerra Sofia Patricia
Chader Gerald J.
Johnson Lincoln V.
Rodriguez Ignacio R.
Steele Fintan R.
Auth Dorothy R.
Feiler William S.
Gucker Stephen
Kunz Gary L.
Morgan & Finnegan L.L.P.
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