Encapsulated Cell Therapy for The Treatment of Epilepsy

Contemporary antiepileptic drugs are ineffective in approximately 30% of the patients. These patients continue to experience seizures and, in many cases, seizures increase in frequency and are associated with significant cognitive decline and psychiatric disorders. The delivery of trophic factors such as glial cell-derived neurotrophic factor (GDNF) to the CNS has tremendous potential for treating a range of diseases including epilepsy. We have recently tested a clinically-validated, implantable cell encapsulation system (EC) that delivers high levels of GDNF in a selective, long-term and stable manner to the epileptogenic area of pilocarpine treated rats. As such, this therapeutic technology platform combines the potency of de novo in situ synthesis of cell-derived GDNF with the safety of an implantable, biocompatible, and retrievable medical device. The de novo synthetized source of very high levels of GDNF in the brain region of interest proved able to significantly reduce generalized seizures frequency, improved cognitive performance and normalized anatomical alterations associated with chronic epilepsy

Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

Delivering glial cell line-derived neurotrophic factor (GDNF) to the brain is a potential treatment for Parkinson'sDisease (PD). Here we use an implantable encapsulated cell technology that uses modified human clonal ARPE-19cells to deliver of GDNF to the brain. In vivostudies demonstrated sustained delivery of GDNF to the rat striatumover 6 months. Anatomical benefits and behavioral efficacy were shown in 6-OHDA lesioned rats where nigraldopaminergic neurons were preserved in neuroprotection studies and dopaminergicfibers were restored inneurorecovery studies. When larger, clinical-sized devices were implanted for 3 months into the putamen ofG\u20acottingen minipigs, GDNF was widely distributed throughout the putamen and caudate producing a significantupregulation of tyrosine hydroxylase immunohistochemistry. These results are thefirst to provide clear evidencethat implantation of encapsulated GDNF-secreting cells deliver efficacious and biologically relevant amounts ofGDNF in a sustained and targeted manner that is scalable to treat the large putamen in patients with Parkinson'sdiseas

Implantable bioartificial hybrids for targeted therapy in the central nervous system

The rapid increase in the understanding of the CNS at the molecular level has brought us new knowledge and tools to attempt to locally manipulate the CNS microenviron- ment and thereby introduce reparative and ablative molecular therapeutic approaches in focal areas of the CNS. This targeted therapy will require neurosurgical intervention for the injection and implantation of cells, viral vectors, or drug release systems. In this thesis, the development of two hybrid systems are presented. One is based on implantable polymeric controlled-release devices that release neurotransmitters, neural growth factors, or synthetic drugs. The other is based on implantable macrocapsules housing neurosecretory cells that are protected from immune rejection. In paper I, the polymeric release of DA for the treatment of Parkinson's disease is studied in a 6-OHDA rat model. The results indicate that direct intrastriatal DA release and diffusion can alleviate Parkinsonian behavior. In paper II, the release of NGF in a rat Alzheimer's disease model is discussed, and the regeneration of cholinergic neurons shown. Paper III describes the development of a co-extrusion method to encapsulate living cells during phase-inversion of a polymeric solution. The use of these capsules for long- term release is discussed. In paper IV, the controlled-release of Amsacrine for glioma therapy is presented. Both histological tumor regression and increased survival are demonstrated in a malignant glioma rat model. In paper V, this system is applied in a human pilot patient and more recent patient data are discussed in conjunction. Paper VI concerns the development of methods to measure bioactivity of released protein factors with results showing that PC12 cells react to GDNF in specific manners. In paper VII the use of the polymeric system to deliver EGF and bFGF for the recruitment of neural stem-cells is investigated. Results show that both EGF and bFGF stimulate striatal progenitor cells but in different manners. The thesis concludes with a general discussion regarding drug transport issues in the brain and a discussion regarding the future of bioartificial hybrids for targeted CNS therapy

Encapsulated cell biodelivery of GDNF: A novel clinical strategy for neuroprotection and neuroregeneration in Parkinson's disease?

The main pathology underlying disease symptoms in Parkinson's disease (PD) is a progressive degeneration of nigrostriatal dopamine (DA) neurons. No effective disease-modifying treatment currently exists. Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective and neuroregenerative effects and it enhances dopaminergic function in animal models of PD. These findings raise the possibility that intrastriatal administration of GDNF might be developed into a new clinical strategy for functional preservation and restoration also in PD patients. Gene therapy is a novel toot to increase local levels of GDNF. Transplantation of encapsulated, GDNF-secreting cells is one strategy for ex vivo cell-based gene delivery which has the advantage to allow for removal of the cells if untoward effects occur. Here we summarize studies with such cells in animals, and discuss the results from previous trials with GDNF in PD patients and their implications for the further development of neuroprotective/neuroregenerative therapies. Finally, we describe the different scientific and regulatory issues that need to be addressed in order to reach the clinic and start the first trial in patients. (c) 2007 Elsevier Inc. All rights reserved

Encapsulated cell therapy in a rat model of epilepsy: long-term, stable, and efficacious targeting of the hippocampus with GDNF

Contemporary antiepileptic drugs are ineffective in approximately 30% of the patients. These patients continue to experience seizures and, in many cases, their seizures increase in frequency and become associated with significant cognitive decline and psychiatric disorders (Klein et al., 2018). The delivery of trophic factors such as glial cell-derived neurotrophic factor (GDNF) to the CNS has tremendous potential for treating a range of diseases including epilepsy. We have developed a clinically-validated, implantable cell encapsulation system (EC) that delivers high levels of GDNF in a selective, long-term and stable manner to the epileptogenic area. As such, this therapeutic technology platform combines the potency of de novo in situ synthesis of cell-derived GDNF with the safety of an implantable, biocompatible, and retrievable medical device. This approach is based on enclosing ARPE-19 cells genetically modified to secrete GDNF in an immunoprotective membrane before transplantation (Fjord-Larsen et al., 2012; Emerich et al., 2014). Initial studies confirmed the long-term (24 weeks) and targeted delivery of GDNF to the rat hippocampus. In subsequent studies, pilocarpine-treated rats, while experiencing spontaneous recurring seizures, received bilateral implants of EC-GDNF devices into the ventral hippocampus. While the number of seizures continued unimpeded in control rats, treatment with EC-GDNF devices reduced seizures by approximately 80% within 2 weeks and by more than 90% within 3 months. These effects persisted even after device retrieval, suggesting potential disease-modifying benefits. Because neuropsychological impairment is a critical co-morbidity of chronic epilepsy we investigated the effects of EC-GDNF on the nature and time course of anxiety-like behaviours and cognitive impairments occurring in pilocarpine treated rats. Importantly, treatment with EC-GDNF maintained normal learning and memory capabilities and normal anxiety-like behaviour. These neurological benefits were associated with the normalization of several anatomical alterations accompanying chronic epilepsy, including preventing hippocampal atrophy, cell degeneration, loss of parvalbumin positive interneurons, and abnormal neurogenesis (Paolone et al., under revision). These studies consistently demonstrated that encapsulated GDNF-secreting cells produce long-term and robust elevations in hippocampal GDNF that are well-tolerated, efficacious and perhaps disease modifying across a spectrum of epilepsy-relevant neurological measures. This approach represents a potentially novel and effective treatment for epilepsy

Leukemia inhibitory factor favours neurogenic differentiation of long-term propagated human midbrain precursor cells

There is a lot of excitement about the potential use of multipotent neural stem cells for the treatment of neurodegenerative diseases. However, the strategy is compromised by the general loss of multipotency and ability to generate neurons after long-term in vitro propagation. In the present study, human embryonic (5 weeks post-conception) ventral mesencephalic (VM) precursor cells were propagated as neural tissue-spheres (NTS) in epidermal growth factor (EGF; 20 ng/ml) and fibroblast growth factor 2 (FGF2; 20 ng/ml). After more than 325 days, the NTS were transferred to media containing either EGF+FGF2, EGF+FGF2+heparin or leukemia inhibitory factor (LIF; 10 ng/ml)+FGF2+heparin. Cultures were subsequently propagated for more than 180 days with NTS analyzed at various time-points. Our data show for the first time that human VM neural precursor cells can be long-term propagated as NTS in the presence of EGF and FGF2. A positive effect of heparin was found only after 150 days of treatment. After switching into different media, only NTS exposed to LIF contained numerous cells positive for markers of newly formed neurons. Besides of demonstrating the ability of human VM NTS to be long-term propagated, our study also suggests that LIF favours neurogenic differentiation of human VM precursor cells

Encapsulated galanin-producing cells attenuate focal epileptic seizures in the hippocampus.

Encapsulated cell biodelivery (ECB) is a relatively safe approach, since the devices can be removed in the event of adverse effects. The main objectives of the present study were to evaluate whether ECB could be a viable alternative of cell therapy for epilepsy. We therefore developed a human cell line producing galanin, a neuropeptide that has been shown to exert inhibitory effects on seizures, most likely acting via decreasing glutamate release from excitatory synapses. To explore whether ECB of genetically modified galanin-producing human cell line could provide seizure-suppressant effects, and test possible translational prospect for clinical application, we implanted ECB devices bilaterally into the hippocampus of rats subjected to rapid kindling, a model for recurrent temporal lobe seizures

Long-term, targeted delivery of GDNF from encapsulated cells is neuroprotective and reduces seizures in the pilocarpine model of epilepsy

Neurotrophic factors are candidates for treating epilepsy but their development has been hampered by difficulties in achieving stable and targeted delivery of efficacious concentrations within the desired brain region. We have developed an encapsulated cell technology that overcomes these obstacles by providing a targeted, continuous, de novo synthesized source of high levels of neurotrophic molecules from human clonal ARPE-19 cells encapsulated into hollow fiber membranes. Here we illustrate the potential of this approach for delivering glial cell line-derived neurotrophic factor (GDNF) directly to the hippocampus of epileptic rats. In vivo studies demonstrated that bilateral intrahippocampal implants continued to secrete GDNF that produced high hippocampal GDNF tissue levels in a long-term manner. Identical implants robustly reduced seizure frequency in the pilocarpine model. Seizures were reduced rapidly and this effect increased in magnitude over 3 months, ultimately leading to a reduction of seizures by 93%. This effect persisted even after device removal, suggesting potential disease-modifying benefits. Importantly, seizure reduction was associated with normalized changes in anxiety and improved cognitive performance. Immunohistochemical analyses revealed that the neurological benefits of GDNF were associated with the normalization of anatomical alterations accompanying chronic epilepsy, including hippocampal atrophy, cell degeneration, loss of parvalbumin positive interneurons, and abnormal neurogenesis. These effects were associated with activation of GDNF receptors. All in all, these results support the concept that implantation of encapsulated GDNF-secreting cells can deliver GDNF in a sustained, targeted, and efficacious manner, paving the way for continuing pre-clinical evaluation and eventual clinical translation of this approach for epilepsy.SIGNIFICANCE STATEMENTEpilepsy is one of the most common neurological conditions, affecting millions of individuals of all ages. These patients experience debilitating seizures that frequently increase over time and can associate with significant cognitive decline and psychiatric disorders that are generally poorly controlled by pharmacotherapy. We have developed a clinically-validated, implantable cell encapsulation system that delivers high and consistent levels of GDNF directly to the brain. In epileptic animals, this system produced a progressive and permanent reduction (>90%) in seizure frequency. These benefits were accompanied by improvements in cognitive and anxiolytic behavior and the normalization of changes in CNS anatomy that underlie chronic epilepsy. Together, these data suggest a novel means of tackling the frequently intractable neurological consequences of this devastating disorder

Cholinergic Profiles In The Goettingen Miniature Pig (Sus Scrofa Domesticus) Brain

Central cholinergic structures within the brain of the even-toed hoofed Goettingen miniature domestic pig (Sus scrofa domesticus) were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) and the low-affinity neurotrophin receptor, p75NTR. ChAT-immunoreactive (-ir) perikarya were seen in the olfactory tubercle, striatum, medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis of Meynert, medial habenular nucleus, zona incerta, neurosecretory arcuate nucleus, cranial motor nuclei III and IV, Edinger-Westphal nucleus, parabigeminal nucleus, pedunculopontine nucleus, and laterodorsal tegmental nucleus. Cholinergic ChAT-ir neurons were also found within transitional cortical areas (insular, cingulate, and piriform cortices) and hippocampus proper. ChAT-ir fibers were seen throughout the dentate gyrus and hippocampus, in the mediodorsal, laterodorsal, anteroventral, and parateanial thalamic nuclei, the fasciculus retroflexus of Meynert, basolateral and basomedial amygdaloid nuclei, anterior pretectal and interpeduncular nuclei, as well as select laminae of the superior colliculus. Double immunofluorescence demonstrated that virtually all ChAT-ir basal forebrain neurons were also p75NTR-positive. The present findings indicate that the central cholinergic system in the miniature pig is similar to other mammalian species. Therefore, the miniature pig may be an appropriate animal model for preclinical studies of neurodegenerative diseases where the cholinergic system is compromised. J. Comp. Neurol. 525:553–573, 2017. © 2016 Wiley Periodicals, Inc

Microglia Impairs Proliferation and Induces Senescence In-Vitro in NGF Releasing Cells Used in Encapsulated Cell Biodelivery for Alzheimer’s Disease Therapy

There is no cure yet available for Alzheimer’s disease (AD). We recently optimized encapsulated cell biodelivery (ECB) devices releasing human mature nerve growth factor (hmNGF), termed ECB-NGF, to the basal forebrain of AD patients. The ECB-NGF delivery resulted in increased CSF cholinergic markers, improved glucose metabolism, and positive effects on cognition in AD patients. However, some ECB-NGF implants showed altered hmNGF release post-explantation. To optimize the ECB-NGF platform for future therapeutic purposes, we initiated in-vitro optimization studies by exposing ECB-NGF devices to physiological factors present within the AD brain. We report here that microglia cells can impair hmNGF release from ECB-NGF devices in-vitro, which can be reversed by transferring the devices to fresh culture medium. Further, we exposed the hmNGF secreting human ARPE-19 cell line (NGC0211) to microglia (HMC3) conditioned medium (MCM; untreated or treated with IL-1β/IFNγ/Aβ40/Aβ42), and evaluated biochemical stress markers (ROS, GSH, ΔΨm, and Alamar Blue assay), cell death indicators (Annexin-V/PI), cell proliferation (CFSE retention and Ki67) and senescence markers (SA-β-gal) in NGC0211 cells. MCMs from activated microglia reduced cell proliferation and induced cell senescence in NGC0211 cells, which otherwise resist biochemical alterations and cell death. These data indicate a critical but reversible impact of activated microglia on NGC0211 cells