Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1998-06-12
2001-05-15
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S366000, C435S377000, C435S455000
Reexamination Certificate
active
06232119
ABSTRACT:
BACKGROUND OF THE INVENTION
The replacement of dopamine (DA) by fetal mesencephalic tissues or DA-cell transplant appears to be a rational choice for the treatment of patients with advanced Parkinson's disease. Fetal mesencephalic tissues have been used extensively in animal models of Parkinson's disease See, for example, Björklund, A. et al. (1979)
Brain Res.
177, 555-560; Perlow, M. F. et al. (1979)
Science
204,643-647; Bakay, R. A. E. et al. (1985)
Appl. Neurophysiol
48, 358-361; Freed, C. R. et al. (1988) in
Pharmacology and Functional Regulation of Dopaminergic Neurons,
eds. Beart, P. M., Woodruff, G. and Jackson, D. M. (Macmillan Press, London) pp. 353-360; Dunnett, S. et al. (1991) in
Intracerebral Transplantation in Movement Disorders,
eds. Lindvall, O., Björkland, A. and Widner, H. (Elsevier, Amsterdam) pp 27-et seq; Freed, W. J et al. (1990)
Exper. Neurol.
110, 139-166; and Kordower, J. H. et al. (1994) in
Neural Transplantation, CNS Neuronal Injury and Regeneration,
eds. Marwah, J., Titelbaum, H. and Prasad, K. N. (CRC Press, Fla.) pp. 17-37. These studies show varying levels of improvement of neurological deficits. The use of such tissues in advanced Parkinson's disease cases also produced variable results as reported in Freed, C. R. et al. (1990)
Arch. Neurol.
47, 505-512; Lindvall,
0
. et al, (1992)
Arch. Neurol.
31, 155-165; Madrazo, I. et al. (1990)
Arch Neurol.
47, 1281-1285; and Hitchcock, E. (1992) in
Recovery from Brain Damage,
eds. Rose, F. D. and Johnson, D. A. (Plenum Press, N.Y.) pp. 67-78. A double-blind study with placebo control to evaluate the efficacy of fetal tissue in the treatment of neurological symptoms in patients with advanced Parkinsonism is in progress. Apart from the issue of efficacy of allogeneic fetal central nervous system (CNS) tissue in neural transplants, the use of fetal tissue is limited by ethical, legal, tissue availability, survivability and some inherent biological problems. The latter includes the presence of donor-antigen presenting cells which may induce rejection of allogeneic grafted tissues. See Freed, C. R. et al. (1990)
Arch. Neurol.
47,505-512; Lindvall, O. et al. (1992)
Arch. Neurol.
31, 155-165; Madrazo, I. et al. (1990)
Arch. Neurol.
47, 1281-1285; Hitchcock, E. (1992) in
Recovery from Brain Damage,
eds. Rose, F. D. and Johnson, D. A. (Plenum Press, N.Y.) pp. 67-78. Another biological problem is heterogeneity within the population of grafted cells. In order to overcome the above difficulties, the establishment of homogeneous populations of DA—producing neurons in vitro, free of antigen presenting cells, would be invaluable. Clonal lines of transformed (tumorigenic) DA-producing neurons, such as those described in Augusti-Tocco, G et al. (1969)
Proc. Natl. Acad. Sci. USA
64, 311-315; Prasad, K. N. et al. (1973)
Nature New Biol.
241, 117-119; Tumilowicz, J. J. et al. (1970)
Cancer Research
30, 2110-2118; Schubert, D. et al. (1974)
Nature
249, 224-227; are available and have been used in neural transplant studies, but such cells produce tumors following transplantation; therefore, they may not be useful until 100% of them are terminally differentiated prior to grafting. See Freed, W. J. et al. (1986)
Exp. Brain Res.
63, 557-566; and Kordower, J. H. et al. (1987)
Brain Res.
417, 85-et seq.
Recently, we have reported the production of 100% terminally differentiated murine neuroblastoma (NBP
2
) cells in culture. See Prasad, K. N. et al. (1994)
Restor. Neurol. Neurosci.
7, 13-19. However, the efficacy of these cells in improving the neurological deficit in the rat model of Parkinson's disease is minimal at 30 days after transplantation. See Adams, F. S. et al. (1996)
Neurochemical Research
21, 619-627. In an effort to establish another source of DA-producing cells, spontaneously or genetically engineered immortalized cells, which include TH-producing fibroblasts, dopamine—producing SV40 LTa gene induced transformed cells at permissive temperature and multipotent neural cells, are now available. See Rosenberg, M. B. et al. (1988)
Science
242, 1575-1579; Anton, R. et al. (1994)
Exp. Neurology
127, 207-218; and Ryder, E. F. et al. (1990)
J Neurobiol.
21, 365-375. The grafting of these cells in animal models of Parkinson's disease has produced variable improvements in neurological deficits when assayed within 3 months of transplant. Recently, we have established an immortalized clone of rat DA—producing neurons (1RB
3
AN
27
) by transfecting fetal mesencephalon cells with the plasmid vector pSV
3
neo
, which carries the large T-antigen gene from SV40 virus. See Prasad, K. N. et al. (1994)
In Vitro Cell. Dev. Biol.
30A, 596-603; Adams, F. S. et al. (1996)
Neuro Chem Res.
21, 619-627. When these cells or differentiated NBP
2
cells were grafted into the striata of 6-hydroxydopamine (6-OHDA) lesioned rats, the methamphetanine-induced turning rate was reduced in about 50% of transplanted animals at 30 days after transplantation; and there was no evidence of rejection of allogeneic cells nor tumor formation at this time. Because this observation period was considered too short for determination of the efficacy of transplanted cells in improving the neurological deficits in lesioned rats, we extended the period of observation to 6 months. In addition, we compared the relative efficacy of undifferentiated and differentiated 1RB
3
AN
27
cells, at improving the neurological deficits in the rat model of Parkinson's disease.
SUMMARY OF THE INVENTION
The present invention teaches improvement of neurological deficits in 6-hydroxy-dopamine lesioned rats following transplantation with allogeneic SV40 large T-antigen gene-induced immortalized dopamine cells.
The replacement of dopamine (DA) by DA neuron transplants in the treatment of advanced Parkinson's disease (PD) is a rational approach. Because of limitations associated with fetal tissue transplants, a clone (1RB
3
AN
27
) of SV40-large T antigen (LTa) gene-induced immortalized rat DA neurons and a line of immortalized human DA neurons were utilized in this study. These allogeneic immortalized dopamine neurons, when grafted into striata of normal rats, did not divide, did not form tumors, did not produce LTa, did not extend neurites to host neurons and were not rejected, for as long as 13 months after transplantation. Grafted cells when recultured in vitro resumed cell proliferation and LTa production, suggesting the presence of a LTa gene-inhibiting factor in the brain. The grafting of undifferentiated and differentiated 1RB
3
AN
27
cells or differentiated murine neuroblastoma (NBP
2
) cells into striata of 6-hydroxydopamine (6-OHDA)-lesioned rats (animal model of PD) caused a time-dependent improvement in neurological deficits (reduction in the methamphetamine-induced turning rate). At 3 months after transplantation, 100% of the animals receiving differentiated 1RB
3
AN
27
cells, 63% of the animals receiving undifferentiated 1RB
3
AN
27
cells, 56% of the animals receiving differentiated NBP
2
cells and 0% of the sham-transplanted animals showed improvements in neurological deficits. At 6 months after transplantation, there was a progressive increase in spontaneous recovery in sham-transplanted animals. These results suggest that immortalized DA neurons may hold potential for their use in transplant therapy in advanced PD patients.
It is an object of the present invention to describe a novel human cell line of immortalized neural cells capable of synthesizing dopamine.
It is a further object of the invention to provide a line of non-tumorigenic, non-immunogenic immortalized human brain cells capable of producing dopamine.
It is a further object of the invention to provide a method of making an immortalized human cell line containing cells capable of producing dopamine, where the cells are non-tumorigenic and non-immunogenic.
It is a further object of the invention to provide a method for treating a neurological deficit characterized at least in part by a localized deficiency of dopamine
Freed Curt R.
Prasad Kedar N.
Crouch Deborah
Greenlee Winner and Sullivan P.C.
Nguyen Quang
University Technology Corporation
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