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Delivery of neurotrophic factors to the CNS using encapsulated cells: Developing treatments for neurodegenerative diseases
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Cell Transplantation, Vo!' 4, Supp!. I, pp. S27-S28, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0963-6897/95 $9.50 + .00
Pergamon 0963-6897(94)00069-7
Original Contribution DELIVERY OF NEUROTROPHIC FACTORS TO THE CNS USING ENCAPSULATED CELLS: DEVELOPING TREATMENTS FOR NEURODEGENERATIVE DISEASES JOSEPH P. HAMMANG,*1 DWAINE F. EMERICH,* SHELLEY R. WINN,* ALICE LEE,* MARK D. LINDNER,* FRANK T. GENTILE,* EDWARD J. DOHERTY,* JEFFREY H. KORDOWER,t AND E. EDWARD BAETGE* *CytoTherapeutics, Inc., 2 Richmond Square, Providence, RI 02906 USA and t Department of Neurological Sciences and Rush Alzheimer's Disease Center, Rush-Presbyterian Medical Center, Chicago, IL 60612 USA
factors using bioassays or ELISA. BHK-control or BHK cells secreting NGF or GDNF were encapsulated in poly(acrylonitrile-co-vinyl chloride) hollow fiber membranes and were held in vitro for 1-4 weeks. Prior to implantation, individual devices were also characterized for growth factor release as above.
BACKGROUND
Protein neurotrophic factors are emerging as potential treatments for a number of chronic neurodegenerative diseases. Delivery of neurotrophic factors to the CNS stably and over the long-term is one of the largest obstacles to the development of effective therapies for these important disorders. The chemical modification of some of these factors may eventually prove effective, however, it remains unclear how readily this strategy will provide new therapies. A number of other approaches are being contemplated, including implantable, growth factor-secreting pumps, implantation of genetically modified cell lines and in situ modification of specific CNS cell populations using viral vector systems. However, all of these approaches suffer from inherent inefficiency or safety issues. The encapsulation of cell lines genetically modified to secrete neurotrophic factors and then transplanted to specific sites within the CNS may provide an alternative and viable approach to treating CNS diseases (3).
RESULTS
NGF - Basal forebrain cholinergic neurons degenerate in several human dementing illnesses including Alzheimer's disease (AD). NGF has potent targetderived trophic and tropic effects upon cholinergic basal forebrain neurons and may represent a useful treatment strategy for AD and/or other diseases characterized by basal forebrain-mediated cholinergic deficits. BHK cell-containing devices were grafted into the lateral ventricle of four cynomolgus monkeys immediately following a unilateral transection of the fornix (1). Three control monkeys received non-hNGF-secreting cells and one monkey received a fornix transection only. Control monkeys displayed extensive losses of choline acetyltransferase and p75 NGF receptor-immunoreactive neurons within the medial septum (MS; 53070 and 54070) and vertical limb of the diagonal band (VLDB; 21070 and 30070) ipsilateral to the lesion. In contrast, monkeys receiving implants of BHK-hNGF cells exhibited a modest loss of cholinergic neurons within the MS (19070 and 20070) and VLDB (7070) together with a dense sprouting of cholinergic fibers within the septum. Retrieved capsules contained numerous viable cells which continued to produce hNGF.
METHODS
The cDNA clones for nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) were cloned into a dihydrofolate reductase-based expression vector (pNUT). Baby hamster kidney fibroblasts (BHK) were transfected with the expression vectors (pNUT-NGF and pNUT-GDNF) and then selected and amplified in methotrexate. The cell lines were assayed for the expression of each of the growth ACCEPTED
'To whom correspondence should be addressed.
7/11/94. S27
S28
Cell Transplantation. Volume 4, Supp!. 1, 1995
GDNF - Parkinson's disease is a progressive neurodegenerative disorder of unknown etiology in which midbrain dopaminergic neurons are gradually lost, leading to movement disorders and eventually death. Recently, a new growth factor, GDNF has been described that exhibits an apparent trophic activity for midbrain dopaminergic neurons in vitro (2). To determine whether GDNF has any effect on dopaminergic function in vivo, two cell lines (sense, antisense) or untransfected BHK cells were encapsulated in immunoisolatory polymeric devices and implanted unilaterally into the striatum of normal Lewis rats. In those animals receiving GDNF, amphetamine-induced asymmetries were apparent. No such asymmetry was seen in the control animals. Histological examination revealed striatal-specific (TH-positive) dopaminergic fiber infiltration into the wall of the GDNF-releasing device. Our preliminary data suggests that GDNF released in a chronic fashion in the rat striatum can influence dopaminergic activity in the nigro-striatal system. Further experiments are needed to determine the extent of GDNF activity in vivo and whether GDNF is protective for midbrain dopaminergic neurons in lesion paradigms. CONCLUSION
The combination of cell lines that have been genetically engineered to secrete neurotrophic factors and
the polymeric membrane-encapsulation technology provides a powerful approach for the treatment of a number of human eNS diseases. Using this system we have successfully shown that factors such as NGF, chronically released in a site-specific fashion result in dramatic neuronal sparing with as little as pg/day levels of NGF. Therapies using this technology may prove advantageous in that secreted proteins may be administered well below the toxic threshold. We believe that this technology holds great promise for the delivery of neurotrophic factors to the central nervous system. REFERENCES
1. Emerich, D.E; Winn, S.R.; Harper, J.; Hammang, J.P.; Baetge, E.E.; Kordower, J.H. Implants of polymerencapsulated human NGF-secreting cells in the nonhuman primate: Rescue and sprouting of degenerating cholinergic basal forebrain neurons. J. Compo Neurol. 349:148164; 1994. 2. Lin, L.-E H.; Doherty, D.H.; Lile, J.D.; Bektesh, S.; Collins, E GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130-1132; 1993. 3. Winn, S.R.; Hammang, J.P.; Emerich, D.E; Lee, A.; Palmiter, R.D.; Baetge, E.E. Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival ofaxotomized septal cholinergic neurons. Proc. Natl. Acad. Sci. USA 91 :2324-2328; 1994.
Cell Transplantation, Vo!' 4, Supp!. I, pp. S27-S28, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0963-6897/95 $9.50 + .00
Pergamon 0963-6897(94)00069-7
Original Contribution DELIVERY OF NEUROTROPHIC FACTORS TO THE CNS USING ENCAPSULATED CELLS: DEVELOPING TREATMENTS FOR NEURODEGENERATIVE DISEASES JOSEPH P. HAMMANG,*1 DWAINE F. EMERICH,* SHELLEY R. WINN,* ALICE LEE,* MARK D. LINDNER,* FRANK T. GENTILE,* EDWARD J. DOHERTY,* JEFFREY H. KORDOWER,t AND E. EDWARD BAETGE* *CytoTherapeutics, Inc., 2 Richmond Square, Providence, RI 02906 USA and t Department of Neurological Sciences and Rush Alzheimer's Disease Center, Rush-Presbyterian Medical Center, Chicago, IL 60612 USA
factors using bioassays or ELISA. BHK-control or BHK cells secreting NGF or GDNF were encapsulated in poly(acrylonitrile-co-vinyl chloride) hollow fiber membranes and were held in vitro for 1-4 weeks. Prior to implantation, individual devices were also characterized for growth factor release as above.
BACKGROUND
Protein neurotrophic factors are emerging as potential treatments for a number of chronic neurodegenerative diseases. Delivery of neurotrophic factors to the CNS stably and over the long-term is one of the largest obstacles to the development of effective therapies for these important disorders. The chemical modification of some of these factors may eventually prove effective, however, it remains unclear how readily this strategy will provide new therapies. A number of other approaches are being contemplated, including implantable, growth factor-secreting pumps, implantation of genetically modified cell lines and in situ modification of specific CNS cell populations using viral vector systems. However, all of these approaches suffer from inherent inefficiency or safety issues. The encapsulation of cell lines genetically modified to secrete neurotrophic factors and then transplanted to specific sites within the CNS may provide an alternative and viable approach to treating CNS diseases (3).
RESULTS
NGF - Basal forebrain cholinergic neurons degenerate in several human dementing illnesses including Alzheimer's disease (AD). NGF has potent targetderived trophic and tropic effects upon cholinergic basal forebrain neurons and may represent a useful treatment strategy for AD and/or other diseases characterized by basal forebrain-mediated cholinergic deficits. BHK cell-containing devices were grafted into the lateral ventricle of four cynomolgus monkeys immediately following a unilateral transection of the fornix (1). Three control monkeys received non-hNGF-secreting cells and one monkey received a fornix transection only. Control monkeys displayed extensive losses of choline acetyltransferase and p75 NGF receptor-immunoreactive neurons within the medial septum (MS; 53070 and 54070) and vertical limb of the diagonal band (VLDB; 21070 and 30070) ipsilateral to the lesion. In contrast, monkeys receiving implants of BHK-hNGF cells exhibited a modest loss of cholinergic neurons within the MS (19070 and 20070) and VLDB (7070) together with a dense sprouting of cholinergic fibers within the septum. Retrieved capsules contained numerous viable cells which continued to produce hNGF.
METHODS
The cDNA clones for nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) were cloned into a dihydrofolate reductase-based expression vector (pNUT). Baby hamster kidney fibroblasts (BHK) were transfected with the expression vectors (pNUT-NGF and pNUT-GDNF) and then selected and amplified in methotrexate. The cell lines were assayed for the expression of each of the growth ACCEPTED
'To whom correspondence should be addressed.
7/11/94. S27
S28
Cell Transplantation. Volume 4, Supp!. 1, 1995
GDNF - Parkinson's disease is a progressive neurodegenerative disorder of unknown etiology in which midbrain dopaminergic neurons are gradually lost, leading to movement disorders and eventually death. Recently, a new growth factor, GDNF has been described that exhibits an apparent trophic activity for midbrain dopaminergic neurons in vitro (2). To determine whether GDNF has any effect on dopaminergic function in vivo, two cell lines (sense, antisense) or untransfected BHK cells were encapsulated in immunoisolatory polymeric devices and implanted unilaterally into the striatum of normal Lewis rats. In those animals receiving GDNF, amphetamine-induced asymmetries were apparent. No such asymmetry was seen in the control animals. Histological examination revealed striatal-specific (TH-positive) dopaminergic fiber infiltration into the wall of the GDNF-releasing device. Our preliminary data suggests that GDNF released in a chronic fashion in the rat striatum can influence dopaminergic activity in the nigro-striatal system. Further experiments are needed to determine the extent of GDNF activity in vivo and whether GDNF is protective for midbrain dopaminergic neurons in lesion paradigms. CONCLUSION
The combination of cell lines that have been genetically engineered to secrete neurotrophic factors and
the polymeric membrane-encapsulation technology provides a powerful approach for the treatment of a number of human eNS diseases. Using this system we have successfully shown that factors such as NGF, chronically released in a site-specific fashion result in dramatic neuronal sparing with as little as pg/day levels of NGF. Therapies using this technology may prove advantageous in that secreted proteins may be administered well below the toxic threshold. We believe that this technology holds great promise for the delivery of neurotrophic factors to the central nervous system. REFERENCES
1. Emerich, D.E; Winn, S.R.; Harper, J.; Hammang, J.P.; Baetge, E.E.; Kordower, J.H. Implants of polymerencapsulated human NGF-secreting cells in the nonhuman primate: Rescue and sprouting of degenerating cholinergic basal forebrain neurons. J. Compo Neurol. 349:148164; 1994. 2. Lin, L.-E H.; Doherty, D.H.; Lile, J.D.; Bektesh, S.; Collins, E GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130-1132; 1993. 3. Winn, S.R.; Hammang, J.P.; Emerich, D.E; Lee, A.; Palmiter, R.D.; Baetge, E.E. Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival ofaxotomized septal cholinergic neurons. Proc. Natl. Acad. Sci. USA 91 :2324-2328; 1994.