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ell and gene therapies in epilepsy -promising avenues or blind alleys

Cell and gene therapies in epilepsy–promising avenues or blind alleys? Wolfgang Lo¨scher1,2,Manuela Gernert1,2and Uwe Heinemann3

1Department of Pharmacology,Toxicology and Pharmacy,University of Veterinary Medicine Hannover,Bu¨nteweg17, 30559Hannover,Germany

2Center for Systems Neuroscience,Bu¨nteweg17,30559Hannover,Germany

3Johannes Mu¨ller Center of Physiology,Institute of Neurophysiology,Charite′,Universita¨tsmedizin Berlin,Tucholskystrasse, 10117Berlin,Germany

The past decades have brought several advances to the treatment of epilepsy.However,despite the continued development and release of new antiepileptic drugs (AEDs),more than one-third of patients are resistant to pharmacological treatment.Furthermore,current AEDs do not prevent the development and progression of epilepsy.Thus,there is an urgent need to develop new therapies for AED-resistant patients,for prevention of epilepsy in patients at risk and for disease modi?cation. Cell replacement and gene therapies have been pro-posed to offer potential approaches for improvements in therapy,but are such approaches really more prom-ising than new pharmacological strategies?Here we critically review and discuss data from epilepsy models and human tissue studies indicating that cell and gene therapies might provide alternative therapeutic approaches for AED-resistant focal epilepsies and might have antiepileptogenic or disease-modifying potential. However,several crucial issues remain to be resolved to develop cell and gene therapies into effective and safe therapies.

Introduction

The majority of patients with epilepsy suffer from focal (partial)seizures,which begin in one part of the brain and then spread.Temporal lobe epilepsy(TLE),the most common and dif?cult to treat type of partial epilepsy (Box1),is typically associated with pathological altera-tions in the hippocampus and parahippocampal regions that are thought to be causally involved in the processes leading to the clinical symptoms of epilepsy and its comor-bidities[1,2](Figure1).Pathological changes in the hippo-campus of patients with TLE are multiple and relate to structural and cellular reorganization of hippocampal formation,selective neurodegeneration and acquired changes of expression,distribution and function of neu-roactive molecules,neurotransmitter receptors and ion channels underlying modi?ed neuronal excitability[1,2], but involve also alterations in glial function[3],mitochon-dria[4]and the blood–brain barrier[5,6].Models of TLE(Box2)are either based on kindling of limbic struc-tures such as the amygdala or hippocampus or a result of experience of a prolonged status epilepticus(SE) induced by kainic acid,pilocarpine or sustained electrical stimulation which lead,after a latency period,to the expression of spontaneous seizures.Based on experiments in such animal models of TLE and clinical studies,an initial brain-damaging insult is thought to trigger a cas-cade of neurobiological events during the latency period (corresponding to epileptogenesis),which leads to the occurrence of spontaneous seizures and to the diagnosis of epilepsy(Figure1).Innovative treatments may either be targeted to epileptogenesis,the morphological and functional changes leading to epilepsy after an initial brain insult,or to ictogenesis,the processes involved in initiation,propagation and ampli?cation of seizures in the epileptic brain(Figure1).Based on this concept and the limitations of conventional therapies(Box1),partial epi-lepsy such as TLE is a potential target for both cell transplantation and gene therapy.These treatments can be used to directly target seizure foci or seizure propa-gation pathways,which is not possible by systemic admin-istration of antiepileptic drugs(AEDs).The topic of cell and gene therapies for epilepsy has recently been covered in several excellent review articles[7–11],and several aspects of this topic are described in much greater detail in these recent reviews.In the present review,we will prim-arily focus on the antiepileptogenic,anticonvulsant and disease-modifying potential of such therapies(Figure1), whereas readers interested in the use of cell or gene therapies for neuroprotection or structural repair in epi-lepsy are referred to previous reviews dealing with this important aspect[9,12].

Transplantation of fetal cells as a potential therapy for epilepsy

Neural transplantation has traditionally been considered in the context of neurodegenerative disorders of the basal ganglia,such as Parkinson’s disease(PD)and Hunting-ton’s disease,which are characterized pathologically by relatively selective cell loss in the basal ganglia,so that cell replacement through transplantation seems logical [13,14].Initial studies with transplantation of rat fetal neural tissue to the adult rat brain were performed some 30years ago by Bjo¨rklund and colleagues,demonstrating survival and growth of such neurons in the striatum and hippocampus,and functional bene?t in rat models of PD, which subsequently led to clinical trials with transplan-tation of fetal human dopamine neurons to the striatum of patients with severe PD[13,14].

Review

Corresponding author:Lo¨scher,W.(wolfgang.loescher@tiho-hannover.de).

In epilepsy,particularly in TLE,cell transplantation could potentially be of value in four different ways (Figure 1):by repairing the damage in the hippocampus,by counteracting or modifying the development of epi-lepsy,by suppressing seizures in AED-resistant patients with established epilepsy or by counteracting the pro-gression of epilepsy.Starting 20years ago,Bjo

¨rklund,Lindvall and colleagues were the ?rst to evaluate the effects of neural transplantation in epilepsy models [13,15].As in models for PD,initial studies used rat fetal neural tissue (Table 1).

Transplantation of fetal noradrenergic or cholinergic neurons into hippocampus or parahippocampal areas In a series of experiments,neurotoxin-induced lesions of forebrain noradrenergic or cholinergic neurons were used to make animals more epileptogenic,that is,to facilitate the development of kindling,an established model of TLE (Box 2).In such rats,transplantation of rat fetal noradren-ergic or cholinergic neurons to the hippocampus or piri-form cortex/amygdala region was ef?cient in retarding seizure development in the kindling model,suggesting an antiepileptogenic or disease-modifying effect.How-ever,such an effect was not observed after neural trans-plantation of noradrenergic neurons in rats without neurotoxin-induced forebrain lesions (Table 1).Further-more,grafting of noradrenergic neurons after completion of kindling did not induce any anticonvulsant effect,which limits suitability of such grafts for possible clinical applications in TLE.

Box 1.Current status of epilepsy therapy

Epilepsy,which is characterized by periodic and unpredictable occurrence of convulsive or nonconvulsive seizures,is one of the most serious brain disorders,affecting 50–100million people worldwide [78].In a majority of patients,seizures have a focal onset,particularly in the temporal lobe.Temporal lobe epilepsy (TLE)is a common end result of brain-damaging insults with very different etiologies and initial pathologies,such as genetic mal-formation,head trauma,stroke,infection or status epilepticus (SE)[1].Following onset of epileptic seizures,patients are usually treated with antiepileptic or anticonvulsant drugs (AEDs)for symptomatic suppression of recurrent seizures.Various new AEDs with diverse mechanisms of action have been developed over recent decades [79,80].However,despite improved tolerability and lower potential for drug interactions of new AEDs,there has been relatively little improvement in AED efficacy since the introduction of phenobarbi-tal in 1912,so that still more than 30%of epilepsy patients are resistant to AEDs [81]with up to 90%with certain types of focal epilepsies.Furthermore,none of the 20old or new AEDs in clinical use appears to represent a ‘cure’for epilepsy or an efficacious means for preventing epilepsy or its progression after brain insults [76].Failure of past drug developments is likely because of a neurocentric approach neglecting the role of the blood–brain barrier,inflammation,astrocytes,mitochondria and genetic dis-position in the disease.Surgical resection of focal,epileptogenic brain tissue is considered the only available curative treatment for patients with epilepsy,but is restricted to patients with AED-resistant seizures,and most patients need continued treatment with AEDs to remain seizure free after surgery [82],implying that in these patients only drug refractoriness is removed.Furthermore,epilepsy surgery has risks and costs that have to be considered,and most forms of AED-resistant epilepsy are not surgically amenable.The adverse seizure and neurobehavioral prognosis in patients with AED-resistant epilepsy provides the justification to search for innovative

treatments.

Figure 1.Steps in the development and progression of temporal lobe epilepsy and possible therapeutic interventions.The term epileptogenesis includes processes that take place before the first spontaneous seizure occurs to render the epileptic brain susceptible to spontaneous recurrent seizures and processes that intensify seizures and make them more refractory to therapy (progression).The concept illustrated in the figure is based on both experimental and clinical data.Adapted from Lo ¨scher [76].

Transplantation of fetal hippocampal neurons into the hippocampus

In contrast to the data with fetal noradrenergic neurons,transplantation of fetal hippocampal neurons to the hippo-campus after kindling reduced the duration of kindled seizures [16].Kindling is an attractive model for testing anticonvulsant effects of neural grafts,because seizures can be reliably produced by electrical stimulation and thus permit easy quanti?cation (Box 2).However,in the kind-ling model of TLE the hippocampus and parahippocampal regions are largely intact,whereas hippocampal damage occurs in most patients with TLE [1].Furthermore,because the hallmark of epilepsy is spontaneous seizures,post-SE models of TLE,such as the kainate and pilocar-pine models (Box 2),showing both spontaneous seizures and temporal lobe damage as components might be better suited for testing the anticonvulsant potential of trans-planted cells.It was therefore important to study whether transplantation of fetal cells to a damaged hippocampus in such models would also provide antiepileptogenic or antic-onvulsant effects as observed in the kindling model.Furthermore,such evaluation of hippocampal fetal cell

grafts on chronic seizures in rat models of TLE is crucial for potential clinical application of this approach because some studies reported that grafts themselves might gen-erate seizures under certain conditions [17–19].

In the kainate model of TLE,transplantation of hippo-campal or locus coeruleus neurons to the ventricles or hippocampus of rats after a kainate-induced SE signi?-cantly reduced the frequency of spontaneous seizures com-pared to sham controls [20].More recently,Shetty and colleagues studied whether neural grafting could be a restorative therapy for hippocampal lesions and epilepsy [12].Studies on the restitution of the disrupted neural circuitry in the injured adult hippocampus with neural cell grafts are valuable for developing treatment strategies that heal hippocampal damage induced by TLE [21–23].There is evidence that neural grafting with appropriate fetal neurons can partially restore damaged structures and neuronal connectivity in models of TLE and prevent the formation of aberrant circuitry [24].In a series of elegant studies,Shetty and colleagues recently developed pre-incubation strategies to enhance graft survival in epileptic hippocampus and demonstrated that partial structural

Box 2.Rat models of temporal lobe epilepsy used in the evaluation of cell and gene therapies in epilepsy

Research on cell and gene therapies for epilepsy has mainly been performed in the following rodent models:(i)the kindling model of TLE,in which repetitive focal electrical stimulation of regions in the temporal lobe results in seizures that progressively increase in severity (graded from stage 1to 5)and length until the animal is ‘fully kindled,’that is,consistently displays partial and secondarily generalized (stage 5)seizures (Figure I a);(ii)acute seizures (status epilepticus;SE)induced by chemical (kainate,pilocarpine)or electrical means (Figure I b);or,less frequently,(iii)chronically recurring spontaneous seizures devel-oping after a chemically or electrically induced SE (Figure I b).Post-SE

models of TLE,such as the kainate or pilocarpine models,are currently considered to have the greatest parallels with TLE,because of the development of spontaneous recurrent seizures and hippocampal damage reminiscent of hippocampal sclerosis typically seen in human TLE [83].However,for assessing the therapeutic effect of novel treatments,these models necessitate laborious long-term monitoring of spontaneous recurrent seizures,which is not needed in models with induced seizures,such as kindling or chemically induced seizures.As shown in Figure I ,cell grafting or gene transfer can either be performed before or after kindling or SE

induction.

Figure I .

repair of the hippocampus is associated with anticonvul-sant and disease-modifying effects[25,26].Pre-incubation with?broblast growth factor2(FGF-2)and brain-derived neurotrophic factor(BDNF)led to lasting survival and integration of grafted hippocampal cells in the injured hippocampus and dampened abnormal mossy?ber sprout-ing[25].In a subsequent study,Rao et al.[26]examined the effects of fetal cell grafts on spontaneous seizures following grafting into the hippocampi of rats exhibiting kainate-induced chronic TLE(Table1).Grafting of hippocampal fetal cells pre-incubated with either FGF-2or BDNF plus caspase inhibitors(to prevent apoptosis)signi?cantly reduced the frequency and progression of spontaneous seizures,whereas grafts without pre-incubation survived poorly in the epileptic hippocampus and did not affect spontaneous seizures[26].This study demonstrates for the?rst time that appropriately treated grafts have the ability to restrain spontaneous seizures in a prolonged fashion(tested up to2months postgrafting)in a rat model of chronic TLE.The mechanisms of this graft effect are not clear,but might include reconstruction of the disrupted hippocampal circuitry and reversal of aberrant mossy?ber sprouting[26].However,almost complete prevention of hippocampal damage in rat models of TLE by prophylactic treatment with neuroprotective drugs such as valproate or topiramate after SE does not prevent development of epilepsy with spontaneous recurrent seizures[27–29],so mechanisms other than structural repair of the damaged hippocampus might explain the anticonvulsant and dis-ease-modifying effect of cell grafting reported by Shetty’s group[26].

Transplantation of fetal GABAergic neurons into the substantia nigra

Apart from targeting fetal neurons to the temporal lobe,an alternative strategy is to place grafts in regions of critical importance for seizure propagation from the temporal lobe (Figure2).In this respect,the pars reticulata of the sub-stantia nigra(SNr)is of interest,because this region and its related circuits within the basal ganglia are thought to function as a gating mechanism for the generalization of convulsive activity[30–32].Inhibition of nigral efferents

by Abbreviations:ACh,acetylcholine;NE,norepinephrine;SNr,substantia nigra pars reticulata.

microinjection of GABA or GABAergic drugs into SNr has been shown to attenuate or block diverse seizure types in a variety of experimental models of epilepsy [30,31].Based on these ?ndings,intranigral grafts of fetal striatal tissue,which have a demonstrated ability to raise local GABA concentrations in vivo ,have been tested in the kindling model,demonstrating a pronounced anticonvulsant effect (Table 1).However,anticonvulsant activity of such grafts was only transient (Figure 3a),even though the grafts survived and contained GABA-expressing cells for several months [33].

Clinical experience with transplantation of fetal GABAergic neurons in patients with epilepsy

The anticonvulsant effect of GABAergic drugs and our promising data from grafting GABAergic neurons in the kindling model [33]prompted a Food and Drug Adminis-tration (FDA)-approved safety and feasibility study of porcine fetal GABA-producing neural cell xenografts in three patients with AED-resistant partial epilepsy [34,35].Cells were transplanted into the ictal focus via modi?ed depth electrodes.Over the months following transplantation,all patients had an improvement in sei-zure frequency,but enrollment of additional patients was halted by the FDA because of the potential risk for

cross-species infection by retroviruses,which might be latent and lead to disease years after infection.These initial clinical data on neural transplantation in epilepsy are encouraging,but much more research remains to be done to establish whether or not neural transplantation will provide an alternative long-term treatment for TLE.Transplantation of genetically engineered cells (ex vivo gene transfer)as a potential therapy for epilepsy

Gene therapy fundamentally involves the transfer of genetic material to a cell and subsequent expression of the gene product [36].There are two main approaches for gene therapy in the brain,ex vivo and in vivo gene transfer.The ex vivo approach involves genetically engineering cells ex vivo to express the desired ‘therapeutic’gene and then implanting the cells into the target tissue.Experiments using this ex vivo approach in rat models of TLE are summarized in Table 2and discussed in the following.For ex vivo gene transfer,immortalized cell lines are used,thereby overcoming the ethical and practical problems associated with the use of fetal cells for neural grafting.Transplantation of GAD 65-transfected cells

In view of the critical role of impaired GABAergic transmission in epilepsy models such as

kindling

Figure 2.Potential routes of seizure propagation of partial (limbic)and secondarily generalized seizures in temporal lobe epilepsy (TLE)and suitable anatomical targets for therapeutic interventions.In TLE and other types of epilepsy,seizure activity does not spread randomly throughout the brain but rather is generated and propagated by specific anatomical routes [30–32].At least in part,the spread of seizures follows pathways that are also utilized in normal movements.Although seizures can be initiated experimentally by a large number of neuronal manipulations,the behavioral alterations associated with different means of seizure induction are often remarkably similar,suggesting that certain propagation pathways might function as common denominators for the development of certain types of epileptic seizures,independent of the specific pathological condition involved in their induction.The same is true for clinical epilepsy,where different types of brain insults can lead to the same type of epilepsy,for example,TLE (Figure 1).In TLE,seizures emanate from the temporal lobe,most often from the hippocampus,entorhinal cortex or amygdala,which therefore form targets for cell-or gene-based therapies aimed at suppressing seizure initiation.Several regions within the temporal lobe,including the hippocampus and dentate gyrus,the entorhinal cortex,the amygdala and the piriform and perirhinal cortices,form an initiating,epileptic circuit as shown in the figure.Based on experimental evidence from the kindling and other models of TLE,the piriform cortex is critically involved in the amplification and propagation of paroxysmal activity emanating from the amygdala or hippocampus [77],so that the piriform cortex is an interesting target for cell-or gene-based therapies aimed at suppressing seizures.Propagation of seizure activity from the temporal lobe to the cortex,basal ganglia,thalamic nuclei and midbrain/brain stem nuclei results in further generalization of seizures.In this circuit,the substantia nigra pars reticulata (SNr)is of particular interest,because inhibition of GABAergic projection neurons of the SNr,such as by potentiation of the inhibitory GABAergic input from the striatum to the SNr or microinjection of GABAergic drugs into the SNr,has been shown to suppress the propagation of a wide variety of seizure types,independent of their origin in the brain [32].A disturbance of this seizure ‘gating’mechanism of the SNr has been implicated in the pathophysiology of different seizure models,including models of TLE [34].Thus,the SNr is an attractive target for therapeutic intervention by neural grafting or gene transfer.In the figure,red and dark blue lines indicate GABAergic and glutamatergic pathways,respectively;gray lines indicate chemically composite pathways.

[37,38],Gernert et al.[39]studied whether kindling in rats can be affected by transplantation of GABA-releasing neurons to the piriform cortex,a brain region that is crucial for the ampli?cation and propagation of paroxysmal activity in the temporal lobe (Figure 2).For this purpose,Gernert et al.[39]used conditionally immortalized mouse neurons that were genetically engineered by Thompson and colleagues [40]to produce and release high concen-trations of GABA by driving the expression of the GABA-synthesizing enzyme glutamate decarboxylase 65(GAD 65).The transplanted cells showed long-term GAD 65expres-sion in the piriform cortex which was associated with signi?cant anticonvulsant effect (Table 2).In a subsequent study by Thompson [41],transplantation of GAD 65-over-expressing mouse neurons to the dentate gyrus was shown to increase GABA levels in the hippocampus and to retard kindling rate,indicating an antiepileptogenic effect of the graft (Table 2).An interesting feature of the cells used by Thompson [41]is that GABA production was under the tight control of doxycycline,thus making individualized GABA delivery possible.This feature might be very important in all cell-based therapies,given the clinical reports that cell transplants with unregulated transmitter release can cause unintended adverse effects [42,43].In

Figure 3.Anticonvulsant effects obtained by neural transplantation or in vivo gene transfer in rat models of temporal lobe epilepsy (TLE).In (a),rats were amygdala kindled until they exhibited a reproducible seizure threshold (afterdischarge threshold;ADT),followed by bilateral transplantation of fetal GABAergic striatal neurons from rats into substantia nigra pars reticulata (SNr),using a microtransplantation approach with eight injections over the anterior–posterior extension of the SNr per side.Following transplantation,ADT determinations were resumed after 10days (p1)and repeated at weekly intervals.Because of a significant increase in ADT,seven of nine rats did not exhibit any electrographic or behavioral seizure activity after transplantation when stimulated with their individual pretransplantation ADT (P =0.021;indicated by asterisk).No such significant anticonvulsant effect was observed in rats that received microinjections of cell-free medium only (not shown).Furthermore,the ADT was not significantly increased in rats that received microinjections of spinal cord cell preparations instead of GABAergic neurons.The strong anticonvulsant effect of grafts of fetal GABAergic neurons was lost over subsequent weeks,although survival of GABAergic neurons in the grafts was verified 11–12weeks after the transplantation.Data are from Lo ¨scher et al.[33].In (b),a similar approach as described above was used in amygdala-kindled rats,but instead of transplanting fetal striatal neurons from rats,we transplanted immortalized GABAergic striatal neurons (M213–2O)from rats bilaterally into the SNr.Following a recovery period of 9–11days after transplantation,the determination of seizure threshold was resumed (p1)and repeated at weekly intervals.Five of seven rats did not exhibit any electrographic or behavioral seizure activity after transplantation when stimulated with their individual pretransplantation ADT (P =0.023;indicated by asterisk).No such significant anticonvulsant effect was observed in rats that received microinjections of a control cell line consisting of non-GABAergic cells (not shown).The strong anticonvulsant effect of grafts of GABAergic neurons was lost over subsequent weeks,although survival of GABAergic neurons in the grafts was verified 4–8weeks after the transplantation.Data are from Nolte et al.[46].In (c),conditionally immortalized mouse cortical neurons were engineered with GAD 65to produce GABA under the control of doxycycline.These cells were bilaterally transplanted into the SNr of spontaneously seizing rats,45–65days after a pilocarpine-induced status epilepticus (SE).Starting 7–8days after transplantation,spontaneous seizures were recorded by video-electroencephalogram over 3subsequent days.For comparison with the GABAergic grafts,rats were either transplanted with cells engineered to produce b -galactosidase (b -gal)or were treated with doxycycline after transplantation of GABAergic grafts to inhibit GABA synthesis in the engineered cells (GABA +Dox).The GABA group had significantly fewer seizures per day compared with the b -gal group and compared with the GABA +Dox group (P <0.05;indicated by asterisk).It was not examined whether this anticonvulsant effect of the GABAergic graft was persistent.Data are from Thompson and Suchomelova [44].In (d),experiments using an in vivo gene transfer approach are illustrated.For this experiment,adeno-associated virus (AAV)vectors were constructed in which the fibronectin secretory signal sequence (FIB)preceded the coding sequence for galanin (AAV-FIB-GAL)or green fluorescent protein (AAV-FIB-GFP).AAV-FIB-GAL or AAV-FIB-GFP were bilaterally infused in the piriform cortex of rats.Seven days after infusion,the animals received a dose of 10mg/kg kainate and were observed for seizures over 240min.For comparison with the rats that received infusions into the piriform cortex,a naive control group was used.All naive controls and all rats with AAV-FIB-GFP developed stage 3seizures,while only 1of 12rats with AAV-FIB-GAL exhibited a single,brief episode of stage 3seizure behavior over the 240min observation period (P <0.01;indicated by asterisk).The other 11AAV-FIB-GAL-treated rats did not exhibit any behavioral or electrographic seizure activity.It was not examined whether this anticonvulsant effect of AAV-FIB-GAL was persistent.Data are from McCown [64].

study in the pilocarpine model of TLE,Thompson and Suchomelova[44]demonstrated that transplantation of the GAD65-tranfected GABA-producing neurons into the SNr suppresses spontaneous seizures(Figure3c),an important step forward de?ning a clinical potential for this approach in epilepsy.However,the long-term ef?cacy of these cell grafts for reducing chronic seizures was not clear because of analyses of seizures only during the early postgrafting period and poor survival of grafted cells in the host brain.

Transplantation of GAD67-transfected cells

Thompson and coworkers used GAD65-transfected mouse neurons for transplantation into rats,that is,a xenograft approach,which was associated with immune reactions in the transplanted region[41]and might reduce the viability of the graft.Freed and coworkers[45]transfected a rat striatal cell line with human GAD67cDNA using an episomal plas-mid vector based on the Epstein-Barr virus,resulting in enhanced GABA synthesis compared to the parent cell line. Allografting these cells in the SNr of rats followed 8weeks later by injection of kainate resulted in suppression of kainate-induced seizures[45].This prompted us to com-pare the effects of the parent striatal cell line(M213–2O)and the GAD67-transfected cell line(M213–2O CL4)following transplantation to the SNr of fully kindled rats[46].Trans-plantation of the immortalized rat striatal cells,M213–2O,resulted in a transient anticonvulsant effect(Figure3b) similar to that previously seen with fetal striatal neurons in this model(Figure3a).However,transplantation of the GAD67-transfected cells,M213–2O CL4,caused strong tis-sue reactions within the host brain of kindled but not of nonkindled rats,characterized by graft rejection with mas-sive in?ltration of immune and in?ammatory cells and gliosis.We previously found that amygdala kindling induces strong activation of microglia in several brain regions,in-cluding the SNr[47],and such overactivation of microglia is known to contribute to in?ammatory and neurotoxic reac-tions[48].These data demonstrate that the host condition might increase the risk of neural transplantation in epilepsy, even when allografts are used.Indeed,the view that the brain is an absolute‘immunologically privileged site’allow-ing inde?nite survival without rejection of grafts of cells has proven to be wrong[49].Instead,the brain should be regarded as a site where immune responses can occur,albeit in a modi?ed form,and under certain circumstances these are as vigorous as those seen in other peripheral sites,necessitating treatment with immunosuppressive drugs[49].

Transplantation of adenosine-releasing cells

Apart from using GABA-releasing cells for neural trans-plantation,cells have been engineered to release the inhibitory neuromodulator adenosine(Table2).In

one 68

study[50],encapsulated adenosine-releasing?broblasts grafted into the brain ventricles of kindled rats provided nearly complete protection from seizures,which,however, was only transient(Table2).In subsequent studies,the potential antiepileptogenic activity of stem cell-mediated adenosine delivery was evaluated[51].

Transplantation or mobilization of stem cells

In the last few years,grafting stem cells or their derivatives for treating neurodegenerative diseases has received con-siderable interest,and TLE was proposed as a potential clinical target for stem cell-based therapies[10].The pro-spects for neural repair and preventing or suppressing seizures in TLE using exogenously applied stem cells are promising,but experimental studies on the ef?cacy of stem cell grafts for treating TLE are very few hitherto[51–54]. Based on promising data with adenosine-releasing?bro-blasts[50],embryonic mouse stem cells,engineered to release adenosine,were differentiated into neural precursor cells and transplanted to the hippocampus of rats before kindling,which retarded subsequent kindling[51].The authors of this study suggested that implants of embryonic stem cell-derived neural precursor cells might display an enhanced potential for long-term seizure suppression effects compared with previously used fetal or genetically engin-eered cells,which,however,needs to be substantiated.

To date,experimental studies on cell-based therapies of TLE have mostly used cells derived from rodents(Tables1 and2),which is not a suitable approach for clinical appli-cations.Autologous patient-derived cell implants would constitute a major therapeutic advance to avoid both xeno-transplantation and immunosuppression.Ren et al.[55] recently described a novel approach based on lentiviral RNAi-mediated downregulation of adenosine kinase,the major adenosine-removing enzyme,in human mesenchy-mal stem cells,which would be compatible with autologous cell grafting in patients.Transplantation of these cells into hippocampi of mice before kainate resulted in anticonvul-sant and neuroprotective effects[55].However,transplan-tation of human neuronal cells(LBS neurons),which recently have been used for neurotransplantation in patients with stroke[56,57],into the hippocampus after kainate-induced SE in rats did not result in suppression of seizures or improvement of learning de?cits observed in this model[58].

Mobilization of endogenous neural stem cells in the adult brain to produce new neurons in the epileptic hippo-campus is an alternative to grafting of stem cells[10]. However,the production of new neurons in conditions such as TLE might be detrimental,as substantially increased neurogenesis observed in the dentate gyrus after SE has been found to be pathological and to promote abnormal hyperexcitability[59].

A weakness in most previous cell transplantation approaches is that principles which failed in pharmaco-logical treatment studies in patients with pharmacoresis-tant epilepsy were reintroduced in the form of grafted fetal or engineered cells.The advantage of neural grafting might lie in the regional modi?cation and prevention of seizure spread,which cannot be achieved by systemic adminis-tration of AEDs.In vivo gene transfer as a potential therapy for epilepsy The in vivo approach of gene therapy uses recombinant viral vectors,where the gene of interest is transferred to the infected cell,followed by expression of the gene pro-duct.This approach has great value in identifying poten-tial cellular alterations as relevant to epileptogenesis and is thus an important research https://www.wendangku.net/doc/f218885078.html,pared to the ex vivo approach,which is limited by restricted viability of cells,in vivo gene transfer promotes long-term expression of the related proteins[8].Several different viral vectors have been developed for in vivo gene therapy but,at present,the two most promising vectors for CNS gene therapy appear to be adeno-associated virus(AAV)and lentiviral vectors [36].Their advantages include the ability to transduce nondividing cells,a preference for neurons,substantial longevity of gene expression and minimal induction of host immune and in?ammatory responses.The majority of in vivo gene transfer studies in epilepsy have used recombi-nant AAV vectors,because they can ef?ciently express therapeutic genes together with a wide range of regulatory elements,such as cell-speci?c or condition-dependent pro-moters.Although this?eld is still in its infancy,the initial experimental studies in models of epilepsy are promising (Table3).

Galanin gene transfer

In vivo gene therapy approaches to epilepsy,so far,have been focused mainly on transduction of neuropeptide genes such as galanin and neuropeptide Y(NPY),which is based on the anticonvulsant effects of these neuropep-tides and their analogs in models of TLE and their ability to antagonize excitatory glutamatergic neurotrans-mission in the hippocampus[8,60].The viability of this neuropeptide gene transfer approach was demonstrated ?rst by Haberman et al.[61],who not only established the anticonvulsant ef?cacy of galanin gene expression in the hippocampus on electrically induced focal seizures (Table3)but also introduced a novel platform(a consti-tutive secretion approach)designed to circumvent the liabilities of viral vector tropism,as a result of the kind of promoter used,previously described by this group[62]. Although galanin gene transfer to the hippocampus did not suppress kainate-induced seizures,it prevented hippocampal hilar cell loss following these seizures[61]. The lack of anticonvulsant effect against kainate-induced seizures in the study of Haberman et al.[61]might be a result of the unilateral application of the vector,because in a subsequent publication,Lin et al.[63]reported that the bilateral intrahippocampal expression of galanin, mediated by an AAV vector carrying a human galanin cDNA,is effective to decrease both the number and duration of kainate-induced seizures(Table3).In a more recent study,galanin gene transfer to the piriform cortex before administration of kainate produced a powerful anticonvulsant effect on kainate-induced seizures in rats (Figure3d)[64].To examine whether this approach is also effective when gene transfer is conducted after onset of seizures,another group of rats was kindled by daily elec-trical stimulation of the piriform cortex until three con-secutive stage5seizures were elicited,followed by galanin gene transfer to the piriform cortex,which signi?cantly

increased the stimulation current necessary to evoke lim-bic seizure activity[64].

NPY gene transfer

In studies on NPY gene transfer,hippocampal expression of the NPY gene delayed seizure onset and reduced the number and duration of seizures induced by kainate and signi?cantly retarded the rate of kindling epileptogenesis [65].The?rst attempt to evaluate the antiepileptic ef?cacy of gene transfer in a chronic rat model of TLE with spon-taneous recurrent seizures and hippocampal damage was recently reported by Annamaria Vezzani’s group[66].The data indicate that intrahippocampal injection of rAAV-NPY in a rat model of chronic epilepsy,ensuing from electrically induced SE,results in long-lasting NPY over-expression in neurons,decreases spontaneous seizure fre-quency and arrests their progression(Table3).Based on these data,a protocol for NPY gene transfer in epilepsy is under evaluation by the FDA[11].Thus,these studies clearly establish the antiepileptic potential for vector-derived galanin and NPY expression in vivo.GDNF gene transfer

Using a neurotrophic factor-based gene therapy approach, Kanter-Schlifke et al.[67]recently demonstrated that AAV-based overexpression of glial cell line-derived neuro-trophic factor(GDNF)in the hippocampus suppresses generalized seizure activity in the kindling model and in an SE model in rats but did not retard kindling-induced epileptogenesis(Table3).The relatively modest effects seen after GDNF gene transfer suggest that cell trans-plantation might be more favorable than single-neuro-trophic factor delivery for treating chronic epilepsy because grafted neurons that are speci?c to the injured area might secrete a multitude of bene?cial trophic factors, in addition to providing additional synapses and facilitat-ing the repair of disrupted circuits[22–26].

Transfer of GABA A receptor subunit genes

The?rst direct evidence that gene transfer can affect the development of epilepsy was recently reported by Raol et al.

[68],using the pilocarpine model of TLE(Box2).Based on previous experiments indicating that

diminished Abbreviations:AAV,adeno-associated virus;ADT,afterdischarge threshold;GDNF,glial cell line-derived neurotrophic factor;NPY,neuropeptide Y.

expression of the a1subunit of the GABA A receptor in dentate granule cells might contribute to epileptogenesis [69],Raol et al.[68]tested the hypothesis that enhancing a1 levels in the dentate gyrus after SE using viral vector gene transfer can inhibit subsequent development of epilepsy.By using an AAV vector with a novel condition-dependent promoter upregulated after SE,a1expression was increased in the dentate gyrus,resulting in a threefold increase in mean seizure-free time after SE and a60%decrease in the number of rats developing epilepsy(Table3).These?ndings suggest that gene transfer strategies designed to modify abnormal gene expression during the latent period between an initial brain injury known to increase risk of epilepsy, such as SE or severe head trauma(Figure1),and the onset of spontaneous seizures might have therapeutic potential for prevention of epilepsy[68].Furthermore,the study demon-strates that in vivo gene transfer is a suitable tool to evaluate the functional impact of brain insult-induced gene alterations in the epileptogenic process(Figure1),which is important both for understanding the molecular mechan-isms of epileptogenesis and for target identi?cation for antiepileptogenic treatments.

Intranasal gene delivery

One major disadvantage of both neural transplantation and gene transfer approaches for treatment of epilepsy is the need of stereotaxic surgery to locate the cells or viral vectors to the target brain region.This restricts the poten-tial use of cell-or gene-based therapies to patients with pharmacoresistant partial epilepsies as an alternative to resective surgery.It is therefore of major interest that Laing et al.[70]recently demonstrated that intranasal delivery of an antiapoptotic gene(ICP10PK),using a growth-compromised herpes simplex virus(HSV)type2 vector(D RR),prevents kainate-induced seizures and neuronal loss in rats and mice(Table3).After intranasal delivery,the antiapoptotic gene gained rapid access to the hippocampus,apparently through the lateral olfactory tract,with expression detected in the olfactory bulb,piri-form cortex,amygdala,thalamus,hypothalamus and entorhinal cortex.The data are promising,but further studies are needed to explore whether D RR is a potential novel platform for treatment of epilepsy.

Gene transfer into the human CNS

No clinical studies using gene therapy have been per-formed so far in human epilepsy.However,a pioneering study using human epileptogenic hippocampal tissue obtained from epilepsy surgery demonstrated the feasi-bility of using AAV vectors to transfer genes into the human CNS and,in particular,into neurons[71].

Apart from using gene transfer for treating sympto-matic epilepsies such as TLE,gene therapy has previously been discussed as a potential strategy to target the speci?c defects underlying inherited epilepsies[72].However, most of the common inherited epilepsies are not trans-mitted through simple(monogenic)inheritance,so their complex(polygenic)transmission complicates attempts to isolate the speci?c defects and to develop appropriate gene therapies[73].Considering the multitude of molecular changes in acquired epilepsies such as TLE,the in vivo gene transfer approach will teach us which molecular changes are indeed critical for epileptogenesis. Concluding remarks

Numerous studies now have demonstrated that cell and gene therapies in acute and chronic models of epilepsy result in anticonvulsant effects,might be antiepileptogenic and might afford neuroprotection or neural repair.How-ever,before moving from preclinical research to the clinical arena,several concerns have to be addressed.Although substantial seizure suppression can be obtained with cell grafting,the anticonvulsant effect was restricted to a few weeks in most rodent studies in which effects were eval-uated over a longer time period.This might be a con-sequence of restricted cell viability,but also of a decrease in transmitter release by the implanted cells or desensitization of target receptors.It is as yet not clear whether this problem can be resolved by stem cell-derived implants.However,the recent studies by Shetty and col-leagues[25,26]suggest that the problem of restricted graft survival can be resolved by pre-incubation of cells with growth-promoting and apoptosis-inhibiting factors.Stimu-lation of neuron formation from the brain’s own stem cells, that is,neurogenesis,might be an alternative to cell graft-ing,although newly formed neurons might not necessarily promote normal function in the epileptic brain.

Compared to cell-based therapies,in vivo gene transfer using AAV vectors might provide more sustained effects in epilepsy models,but this remains to be demonstrated in animal models of chronic epilepsy,because receptor desen-sitization might occur,depending on the therapeutic gene, as in cell-based approaches.Furthermore,the presence of antibodies to wild-type AAV in the human population could result in inactivation of recombinant AAV vectors used for gene therapy[36,74],but a recent study using AAV-based GAD gene transfer to the subthalamic nucleus of patients with Parkinson’s disease did not indicate that antibody responses to the AAV vector affected the response to gene therapy[75].However,as demonstrated by our recent experiments with transplantation of GAD67-transfected neurons in kindled rats[46],alterations in epileptic tissue before intracerebral cell or gene application might dramatic-ally affect the ef?cacy of such approaches.These problems are under investigation in animal models and we hope can be resolved soon,so it should be possible to realize ef?cacious cell or gene therapies for patients with intractable focal epilepsy and possibly for preventing symptomatic epilep-sies.Only clinical trials will ultimately prove whether cell or gene therapies are more promising than new pharmacologi-cal strategies for treatment of epilepsy. Acknowledgements

We thank Thomas J.McCown and Steven C.Schachter for providing information on some aspects of this review and Annamaria Vezzani for critical comments on an earlier draft of the manuscript.The authors’own studies were supported by grants from the Deutsche Forschungsgemeinschaft(Bonn,Germany).The authors declare that they have no competing?nancial interests.

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