Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-01-22
2001-02-06
Brusca, John S. (Department: 1635)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093200, C435S006120, C435S320100, C435S325000, C435S353000, C435S440000, C536S023100, C536S023500, C536S023700
Reexamination Certificate
active
06183735
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel immortalized cell lines of retinal origin (retinal endothelial and retinal pigmentary epithelial origin) which are capable of being implanted in the retina and of conveying a substance of therapeutic interest into the eye and the central nervous system. Such lines can also serve as a model for studying the blood/central nervous system interfaces.
2. Description of the Background
Both the blood-brain barrier and the blood-retina barrier are important in controlling the passage of substances to and from the neural parenchyma, especially in the maintenance of haemostasis.
In the retina, the blood-retina barrier comprises two different types of cells which are anatomically separate. The retinal vascular endothelium, which supplies the anterior portion of the retina, is currently considered to have an identical structure to the cerebral endothelium (Towler et al., J. Physiol., 1994, 480, 10-11P), whereas the cells of the retinal pigmentary epithelium cover the permeable vessels of the choroidal circulation and form the posterior barrier by means of their tight apical junctions; they are consequently similar to the tight junction epithelial cells of the choroid plexus.
The cerebral and retinal endothelia are of a different nature to the peripheral endothelium and do not serve only to express tight junctions and form a physical barrier. Other properties of the endothelium contribute to the specialized nature of this barrier, particular properties being the distribution and expression of substances such as the glucose transporter (GLUT-1), the transferrin receptor and P-glycoprotein (Pgp), the expression product of the drug resistance gene.
The cerebral and retinal endothelia also differ from the peripheral endothelium in their permeability to the circulating leukocytes.
The in vitro study of the molecular mechanisms of the induction of the endothelial phenotype has the disadvantage of being dependent on the availability, in primary cell cultures, of endothelial cells of cerebral or retinal vessels or pigmentary epithelial cells of the retina.
As far as in vivo transfer is concerned, the use of primary nerve tissues of foetal origin for cellular transplantation in human therapy gives rise to numerous ethical and practical problems; one alternative to this problem is to use primary cell lines of neural origin (for example neurons, glial cells, astrocytes) or non-neural cell cell lines (for example fibroblasts, myoblasts, chromaffin cells of the adrenal medulla, hepatocytes). Although the cell lines of the adrenal medulla, fibroblasts or myoblasts can actually release active substances in vivo, they are not normally present in the central nervous system, but can modify the normal function of the nervous system and cause a rejection reaction.
Because of the heterogeneity of the endothelial cells of different tissues, influenced by the environment of these cells, it was important to be able to have cells adapted to the retinal environment in order to have tools permitting a good morphological and physiological integration of the cells when they are implanted or grafted.
Consequently, the Inventors set themselves the task of providing immortalized cell lines derived from primary cultures of the endothelium of the blood-retina barrier and the retinal pigmentary epithelium of mammals, especially rodents and more particularly rats, which cell lines are better capable of meeting practical needs, especially in that all the lines obtained are stable and have most of the characteristics of the differentiated cells.
SUMMARY OF THE INVENTION
The present invention relates to immortalized mammalian cell lines, characterized in that they are derived from primary cultures of retinal cells selected from the group comprising the primary retinal endothelial cells and the primary retinal epithelial cells, in that they comprise a nucleic acid fragment containing at least one immortalizing fragment of a heat-sensitive viral oncogene, which nucleic acid fragment may be associated with at least one selection gene, and in that they stably exhibit the morphological characteristics and at least the surface antigen expression characteristics of the corresponding primary culture cells.
DETAILED DESCRIPTION OF THE INVENTION
Stability is understood as meaning the maintenance of the morphological and phenotypic characteristics of the immortalized lines for up to at least 30 passages and even up to more than 50 passages.
In one advantageous embodiment of said lines, they are derived from retinal endothelial cells and exhibit at least the morphological characteristics and antigen expression characteristics of the primary culture retinal endothelial cells, namely fusiform morphology, expression of the endothelial tissue markers such as RECA-1, constitutive expression of markers specific for the CNS endothelium, such as at least P-glycoprotein, GLUT-1 and the transferrin receptor, and absence of expression of surface antigens specific for the cerebral endothelial cells, such as the 1A8B antigen.
In another advantageous embodiment of said lines, they are derived from retinal pigmentary epithelial cells and exhibit the morphological characteristics and antigen expression characteristics of the primary culture retinal pigmentary epithelial cells, namely pavement morphology and expression of RET-PE2 and cytokeratins.
Surprisingly, such cells do not differ in their expression of the characteristics of the differentiated retinal endothelial cells or the differentiated retinal pigmentary epithelial cells.
Moreover:
the retinal pigmentary epithelial cells are capable, in vivo, of integrating appropriately into the cytoarchitecture of the retina of a host mammal without proliferating, and of preventing the loss of photoreceptors, especially in rats of the RCS (Royal College of Surgeons) strain, and
the retinal endothelial cells are capable, in vitro, of serving as a model for the blood-retina barrier in the absence or presence of the retinal pigmentary epithelial cells.
In another advantageous embodiment of said lines, the nucleic acid fragment containing at least one immortalizing fragment of an oncogene contains a fragment of a heat-sensitive SV40 T-oncogene.
This gave:
immortalized retinal endothelial cells, called IO/JG2/1, which were fusiform like the primary culture cells and expressed the above-mentioned markers specific for the CNS endothelial cells, as well as the same surface antigens and the same antigens of the major histocompatibility complex as the primary culture retinal endothelial cells, and
immortalized retinal pigmentary epithelial cells, called IO/LD7/4, which were very similar to the primary culture cells; although not pigmented, these cells express the specific RET-PE2 antigen and the cytokeratins.
According to the invention, the immortalized retinal endothelial cells called IO/JG2/1 were deposited under no. I-1695 on Apr. 18th 1996 in the Collection Nationale de Cultures de Micro-organismes held by the Institut Pasteur, 28 rue de Docteur Roux, 75724 PARIS CEDEX 15.
According to the invention, the immortalized retinal pigmentary epithelial cells called IO/LD7/4 were deposited under no. I-1694 on Apr. 18th 1996 in the Collection Nationale de Cultures de Micro-organismes held by the Institut Pasteur.
The present invention further relates to cell lines derived from the immortalized lines as defined above, hereafter called vector cell lines, characterized in that they comprise at least one cell line as defined above, associated with an expression vector comprising a sequence coding for a polypeptide, a protein or a viral vector, optionally associated with at least one selection gene and optionally at least one marker gene, and in that they are capable, in vivo, of integrating into the retina and especially the subretinal space of a host mammal, preventing the loss of photoreceptors and producing said peptide, said protein or said viral vector.
In terms of the present invention, expression vector is understood as meaning
Adamson Peter
Greenwood John
Lund Raymond
Brusca John S.
Elrifi Ivor R.
Mintz, Levin, Cohn, Ferris, Glovsky and Popeo, P. C.
Neurotech SA
Prince John T.
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