Increased Encapsulated Cell Biodelivery of Nerve Growth Factor in the Brain by Transposon-Mediated Gene Transfer

ORIGINAL ARTICLE Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

L Fjord-Larsen,1, P Kusk1, DF Emerich2, C Thanos3, M Torp1, B Bintz3, J Tornøe1, AH Johnsen4 and LU Wahlberg2

Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer’s disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modiﬁed human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to signiﬁcantly higher NGF output, which in both cases correlate with highly improved potency.

Gene Therapy (2012) 19, 1010--1017; doi:10.1038/gt.2011.178; published online 24 November 2011 Keywords: encapsulated cell biodelivery; sleeping beauty transposon; NGF; preclinical validation; cholinergic neurons; rat QA model

INTRODUCTION zoonosis and xenogenic reactions from the secretome, a human Alzheimer’s disease (AD) is a progressive, fatal neurodegenerative cell line is preferred for clinical applications. We have previously disorder characterised by deterioration in cognition and memory, described clinically relevant, NGF-secreting EC Biodelivery devices, progressive impairment in the ability to carry out activities of daily which can be safely implanted in the brain, retrieved and show 15 living and a number of neuropsychiatric symptoms. The degen- long-term function. The clinical product, named NsG0202, eration of cholinergic neurons in the basal forebrain correlates contains NGF-secreting NGC-0295 cells, derived from a human with the cognitive decline in AD patients.1 While current drug retinal pigment epithelial (RPE) cell line, ARPE-19. This sponta- treatments for AD offer transient benefits to some patients, there neously immortalised cell line shows contact-inhibited growth and 16 is an urgent need for improved treatments and, in particular, survives well in nutrient-poor culture environments, making it disease-modifying therapies. A promising therapeutic agent in well suited for encapsulation. A small phase 1b clinical study to this respect is Nerve Growth Factor (NGF), which has shown examine the safety of NsG0202 devices implanted in the basal neuroprotective and regenerative effects on the cholinergic forebrain of patients with mild to moderate AD has shown neurons in the basal forebrain in various animal models, such as promising results (Eriksdotter-Jo¨nhagen et al.; Wahlberg et al., axonal injury, spontaneous age-related atrophy and impaired manuscripts submitted). The successful safety profile in all patients learning and memory in lesioned and aged rats or primates.2--8 and relevant improvement in cognition and biomarkers in some Besides bypassing the blood--brain barrier, a challenge for clinical patients suggest that a dose escalation step is relevant. application is the safe delivery of therapeutic doses of NGF to the To accomplish higher secretion, we have generated regulatory target area, as impaired endogenous transport of NGF to the compliant clonal NGF-secreting ARPE-19 cell lines using the 17 cholinergic neurons is part of the AD pathology.9 Encapsulated sleeping beauty (SB) transposon expression system. This Cell (EC) Biodelivery combines the advantages of gene therapy technology uses an optimised SB transposase (SB100X), which with the safety of a retrievable and replaceable medical device.10 mediates genomic integration of multiple copies of the transgene The implantable EC Biodelivery device containing a genetically inserted between two transposon terminal inverted repeats. The engineered cell line secretes a low dose directly into the relevant preferred sites for genomic SB integration are palindromic AT anatomical region of the brain. The semipermeable membrane repeats, and the insertion site distribution is nearly random. The allows for influx of nutrients and the efflux of the therapeutic vector system is capable of stable gene transfer with long-term protein, and immunologically isolates the host brain from the ECs. gene expression at an efficiency comparable with that of viral As applied to AD, the device contains NGF-secreting cells and is systems.18 From the established clones, we have selected the cell placed in the medial and lateral aspects of the basal forebrain. line NGC0211, showing at least 10-fold higher NGF expression Several studies have shown beneficial effects of encapsulated than cell line NGC-0295, generated using standard transfection NGF-secreting baby hamster kidney cells or rat fibroblasts in techniques. Correct processing of NGF from NGC0211 was animal models with relevance for AD.11 --14 However, to avoid confirmed, and the bioactivities of devices with NGC0211 and

1NsGene A/S, Ballerup, Denmark; 2NsGene Inc., Providence, RI, USA; 3CytoSolv Inc., Providence, RI, USA and 4Department of Clinical Biochemistry, Copenhagen, Denmark. Correspondence: Dr L Fjord-Larsen, NsGene A/S, Baltorpvej 154, Ballerup, DK-2750, Denmark. E-mail: lfl@nsgene.dk Received 11 May 2011; revised 9 September 2011; accepted 10 October 2011; published online 24 November 2011 Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1011 NGC-0295 were validated in preclinical animal studies in normal devices was measured before implantation and after explantation rat brain and in the quinolinic acid (QA) striatal lesion model of (Figure 2a). Release from devices with NGC0211 cells (9.1±0.3 ng neuronal degeneration. per 24 h) was significantly higher than from devices with NGC-0295 cells (1.0±0.1 ng per 24 h) before implantations (t-test, Po0001). After explantation, the measured NGF release was RESULTS around seven times higher for both cell lines. No NGF release was NGC0211 cells secrete high amounts of correctly processed NGF detected from devices with parental ARPE-19 cells (Figure 2a). The transgene copy number in the NGC0211 cell line was Quantifications of NGF immunostainings reflected weak staining determined to be four using a derivative of the splinkerette around NGC-0295 devices and a prominent NGF immunoreactivity technique.19 NGF release from NGC0211 cells was more than covering the main part of the striatum around NGC0211 devices 10-fold higher than NGC-0295, created using standard liposome (Figure 2b). The radius of visible immunoreactive NGF from the transfection techniques (B400 ng 10À6 cells per 24 h versus implant edge was up to 2 mm. Explanted devices contained B30 ng 10À6 cells per 24 h). healthy living cells (Figure 2c). Processing of NGF secreted from NGC0211 cells was investi- In an additional experiment, NGF levels in the tissue surround- gated by western blotting analyses, which showed a monomer of ing devices with NGC0211 cells and NGC-0295 cells, respectively, processed mature NGF with the expected molecular weight of were determined by NGF enzyme-linked immunosorbent assay 13.2 kDa (Figure 1a). Faint bands representing the molecular (ELISA) analyses of homogenised punches, taken at the implant weight of the NGF dimer was also seen. Importantly, no pro-NGF site. Results showed high NGF levels (128±26 pg mgÀ1 tissue) (migrates at 32--34 kDa) was detected. As previously observed for around devices with NGC0211 compared with devices with NGF produced from NGC-0295 cells,15 a small fraction (2--3%) of NGC-0295 cells (10.1±2.5 pg mgÀ1 tissue). partially processed NGF, represented by a double band with To further characterise the increased NGF release measured molecular weight between 21.3 and 29 kDa, was present. As also after explantation, the membranes from 50% of the explanted described for NGC-0295 cells, we observed a higher molecular devices were separated from the cell-containing yarn matrix, weight band (436 kDa) in the NGC0211-produced NGF sample. and the NGF release was determined. Intact explanted devices This band was not recognised by a pro-NGF antibody (data not and devices kept in vitro throughout the experimental period shown) and may represent an aggregated form of NGF, possibly were used as references. In this experiment, NGF release from formed during the processing of samples for SDS-PAGE. In the intact explanted devices was around five times higher than shown experiment, it was not detected in the NGC-0295-produced from the in vitro devices. However, from the explanted devices, a NGF sample. The exact processing of the NGF secreted from high NGF release was measured from the membranes with no NGC0211 cells was checked by high-pressure liquid chromato- cells (55.7±11.9% of release measured from intact explanted graphy (HPLC) purification and MALDI-TOF mass spectrometry. devices) compared with release from membranes taken from the NGF was purified by three HPLC steps and the last step resulted devices kept in vitro (13.8±0.8% of release measured from intact in several discrete ultraviolet peaks. One of these showed high devices). NGF immunoreactivity and also the expected characteristics of mature NGF, as analysed by mass spectrometry and top--down Higher potency of NGC0211 devices in normal rat brain sequencing (Figure 1b). NGF has previously been shown to increase the size of intact as well as degenerating cholinergic neurons.20 --22 Accordingly, larger Increased delivery of NGF from devices with NGC0211 cells in cholinergic neurons, expressing choline acetyltransferase (ChAT), normal rat brain were seen around devices with both of the NGF-producing cell To compare the in vivo delivery of NGF, devices with NGC0211 lines, compared with devices containing the parental ARPE-19 cells, NGC-0295 cells or parental ARPE-19 cells were implanted in cells (Figure 3a). The size of the ChAT-positive neurons in a rat striata and explanted after 2 weeks. NGF release from distance of up to 2 mm from the devices was quantified by image

Figure 1. Characterisation of NGF produced from NGC0211 cells. (a) NGF western blotting of conditioned medium from NGC-0295 and NGC0211 cells. Purified recombinant human NGF (rh NGF) is included as reference. The mature monomer (NGF) is indicated by an arrow. A band, corresponding to the dimer, and the double band, representing intermediate partially processed forms (pp NGF), are also shown by arrows. (b) The immunoreactive peak from the last HPLC purification step analysed by MALDI-TOF mass spectrometry. The average molecular mass was 13 488.0 Da, compared with the theoretical 13.488,4 Da of intact NGF. Top--down sequencing confirmed the residues 10--15 of NGF. In addition, the remaining N-terminal fragment (Ser-Ser-Ser-His-Pro-Ile-Phe-Arg) had the molecular mass 1066.8 Da compared with the theoretical 1066.5 Da.

& 2012 Macmillan Publishers Limited Gene Therapy (2012) 1010 --1017 Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1012

Figure 2. NGF release from devices with parental ARPE-19 cells, NGC-0295 or NGC0211 cells, implanted in normal rat brain for 2 weeks. (a) NGF release measured from devices before and after the 2 weeks in rat brain. Error bars denote s.e.m., n ¼ 8(b) Quantiﬁcation of the NGF immunostaining in tissue surrounding devices. Data are shown as the mean NGF-immunoreactive area per section (±s.e.m., n ¼ 8). Insert shows a brain having a device with NGC-0295 cells in the left side and one with NGC0211 cells in the right side. Scale bar ¼ 2 mm). (c) Hematoxylin- and eosin-stained sections of explanted devices with the three indicated cell types. Scale bar ¼ 300 mm.

Figure 3. Response of cholinergic cells around devices. (a) ChAT-immunoreactive cells (arrows) in the vicinity of implant site (i) of devices with the three cell types. NGF secreted from devices increases cell size. Scale bar ¼ 200 mm. (b) Sprouting of cholinergic (p75NGFR- immunoreactive) ﬁbres (arrows) around the implant site (i) of devices with the three cell types. Scale bar ¼ 500 mm.

Gene Therapy (2012) 1010 --1017 & 2012 Macmillan Publishers Limited Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1013 analyses, which showed a significant effect of NGC-0295 devices Improved protection by NGC0211 cells in QA-lesioned rats (367±19 mm2 per neuron) compared with ARPE-19 devices Next, we wanted to validate the in vivo potency of devices with (298±11 mm2 per neuron). Furthermore, the mean size of NGC-0295 and NGC0211 cells in a model of striatal neurodegene- ChAT-positive neurons in the vicinity of devices with NGC0211 ration, the QA lesion model, in which NGF has previously shown cells (386±12 mm2 per neuron) was significantly larger than around protective effects.23,24 Empty devices and devices with the devices with NGC-0295 cells (Po0.05). There was no difference in parental ARPE-19 cell line were included as controls. Unilateral the number of ChAT-immunoreactive neurons per section striatal implantations of devices were performed 2 weeks before between the treatment groups (P40.05). NGF also induces inducing an excitotoxic lesion by intrastriatal QA injection. The sprouting of cholinergic fibres.4,6 As shown in Figure 3b, we animals were scored in two behavioural tests for motor deficits observed a significant increase in sprouting of p75NGFR- before QA injections and again 2 and 4 weeks after. After the last immunoreactive fibres around devices with NGF-producing cells. test, animals were killed, and histological analyses were performed Quantification by image analysis showed significantly more to examine the cellular responses in the brain. sprouting around devices with NGC0211 cells (2.281±0.142 mm2 Figure 4a shows results from the forelimb placing test, where p75NGFR-immunoreactive area per section) than around devices performance of the unimpaired ipsilateral forelimb was consistent with ARPE-19 cells (0.349±0.027 mm2 per section) and NGC-0295 with normal function with all treatment groups making 49.4 out cells (1.186±0.063 mm2 per section), Po0.05. of 10 correct responses. The QA lesion resulted in marked

Figure 4. Protective effect of devices with NGF-secreting cells in the rat QA lesion model. (a) Forelimb placing test. Data are presented as the mean±s.e.m. Number of correct places out of 10 consecutive trials with the forelimbs ipsilateral and contralateral to the striatum 2 and 4 weeks after the QA lesion, n ¼ 8. The * shows a significant difference (Po0.05) between the indicated treatment groups. (b) Spontaneous forelimb use in the cylinder test. Values displayed represent the percent symmetry as mean contralateral touches/total touches±s.e.m., 2 and 4 weeks after QA lesion (n ¼ 8). The * indicates a significant difference between empty devices and devices with ARPE-19 cells. (c) Weight curves for the four experimental groups. Data are shown as the mean increase±s.e.m. (n ¼ 8), and * indicates a significant difference between the NGC0211 group and the other three groups. (d) Devices with NGC0211 decreases lesion size at the implant site (Bregma 0.0). Selected sections at the indicated coordinates were analysed. Data are shown as the mean lesion area per section ±s.e.m. (n ¼ 8) and * indicates a significant difference from devices with empty cells, ** indicates a difference from devices with ARPE-19 cells and NGC-0295 cells. (e) Quantification of the number of ChAT-immunoreactive neurons up to 2 mm from the implant. Data are shown as the mean number per section ±s.e.m. (n ¼ 48 sections) and * indicates a significant difference between groups with NGF-producing cells and controls.

& 2012 Macmillan Publishers Limited Gene Therapy (2012) 1010 --1017 Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1014 performance deficits in the contralateral side in the control groups devices containing parental ARPE-19 cells. Posterior to the device, with empty devices and devices with the parental cell line. Rats the groups with NGF-producing cells had a significantly smaller having devices with NGF-producing cells showed significantly lesion than animals with empty devices. There was no difference improved performance. After 2 weeks, there was no difference in the lesion size between the groups at the QA injection site or between the devices with NGC0211 and NGC-0295 cells (5.0±0.3 0.5 mm anterior. In accordance with the smaller lesion size, the vs 4.8±0.4), whereas there was a significantly better effect of number of ChAT-positive neurons in a distance of 2 mm from the devices with NGC0211 at 4 weeks after inducing the lesion devices was significantly higher around devices with NGC-0295 (5.1±0.3 vs 4.4±0.3, Po0.05). The results of the spontaneous and NGC0211 cells than around empty devices and devices with forelimb use in the cylinder test were in accordance with those ARPE-19 cells (Figure 4e). No cholinergic sprouting was observed from the placing test (Figure 4b). At both testing points, a in the QA-lesioned brain (data not shown), and the size of prominent decrease in the forepaw use in the limb contralateral to cholinergic neurons around control devices (242±6 mm2 per the injected striatum was observed in the groups with empty neuron) did not differ significantly from the neuronal size around devices (16.4±2.8 and 15.0±2.7% symmetry at 2 and 4 weeks, NGF-secreting devices (241±8 for NGC0211 and 226±5 mm2 per respectively) and devices with ARPE-19 cells (15.0±1.9 and neuron for NGC-0295, P40.05). 14.3±2.6% symmetry at the two time points). Animals implanted In contrast to the data obtained from normal brain, the mean with devices containing NGF-producing cells performed NGF release measured from explanted devices with NGC0211 cells significantly better, with at least 30% symmetry (Po0.05). In decreased compared with the pre-implantation values (from the cylinder test, there was no significant difference between the 13.5±1.5 to 1.0±0.3 ng per 24 h) in the QA-lesioned brains. The groups with NGC-0295 and NGC0211 cells. NGF release from devices with NGC-0295 seemed more stable, but Weight loss is a common observation when injecting QA in rat low (from 1.8±0.4 to 2.5±1.0 ng per 24 h). Accordingly, the NGF striatum.25,26 Though we did not record a QA-induced weight around the devices in the QA-lesioned brains was below the reduction, we observed a significant effect of the implants with detection level for the immunohistochemistry method (data not NGC0211 cells, as the weight increase in the first week after QA shown). Devices with both the parental cells (data not shown) and lesion and the mean weight throughout the study was the NGF-producing clones contained obviously healthy living cells significantly larger for the NGC0211 group than the other three (Figures 5a and b). However, in some devices a fraction of dead experimental groups (Figure 4c). No difference was found cells (without nuclei) was observed. Estimating the cell number of between the mean weight of the two control groups and the explanted NGC0211 and NGC-0295 cells by quantifying the area of NGC-0295 group. nuclei per device section (Figure 5c) showed no significant To visualise NGF-induced neuronal protection against QA difference (P ¼ 0.282). toxicity, brains were immunostained using an antibody against NeuN, expressed in the nuclei of most neuronal cell types in the brain.27 The size of the striatal lesion, devoid of NeuN-positive DISCUSSION neurons, was quantified on coronal sections at four AP In the present study, we have obtained a 10-fold increase in NGF coordinates: þ 1.5, þ 1.0 (QA injection site), 0.0 (device implant) expression from the EC Biodelivery platform cell line, ARPE-19, by and À1.0, relative to Bregma (Figure 4d). At the device implant using the SB transposon technology. The resulting expression level, the lesion area was significantly smaller in the NGC0211 level is also at least 10 times higher than previously described for group than in the other three experimental groups (Po0.05). The NGF-secreting rat fibroblasts used for grafting or encapsula- mean lesion area around devices with NGC-0295 cells was smaller tion.6,11,23 The multiplicity of transgene copies may be the primary than around empty devices but not different from the group with reason for the enhanced expression compared with standard

Figure 5. Haematoxylin- and eosin-stained sections of explanted devices. Representative explanted devices with (a) NGC-0295 cells and (b) NGC0211 cells. Healthy cells with dark-stained nuclei are seen (arrows). Scale bar ¼ 300 mm. (c) Relative survival determined by quantiﬁcation of the total nuclei area in device sections, shown as mean area per section ±s.e.m. (n ¼ 8 devices).

Gene Therapy (2012) 1010 --1017 & 2012 Macmillan Publishers Limited Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1015 transfection techniques. Prevention of transgene downregulation affected by QA concentrations up to 10 mM (data not shown), but could be another factor. Transgenes delivered by non-viral the ECs may be distressed by the QA-induced secondary approaches often lead to concatamer-induced gene silencing by inflammatory responses in the brain. The exact timing of the heterochromatin formation. As the cut and paste mechanism of decrease in NGF release is unknown, but evidently the higher transposon-mediated gene transfer results in a single copy of the levels of NGF present at the time of the QA injection were gene per insertion locus, this downregulation is avoided. sufficient for a significant and lasting protection against the Furthermore, results from SB transposon-mediated gene transfer excitotoxic insult. In contrast to the findings in normal rat brain, in HeLa cells suggest that inactive heterochromatin regions are cholinergic sprouting or hypertrophy around devices with NGF- rarely targeted.28 secreting cells was not observed in the QA-lesioned brain. This is NGF secretion as correctly processed bioactive molecules from in agreement with the subsequently lower NGF levels, and that NGC0211 cells was confirmed by mass spectrometry, and we some NGF effects are not maintained after abolishing treat- found that the differences in the NGF release in vitro correlated ment.31,32 The diseased AD brain environment also shows well with NGF concentrations measured in tissue surrounding increased inflammatory responses and oxidative stress,33 --35 which devices with the two cell lines. Quantification of NGF immuno might affect cells negatively in implanted devices. However, the stainings gave an even higher difference (B50 times). This result, inflammatory environment in the AD brain is less severe than in however, is likely to reflect that the NGF concentrations around the QA-lesioned rat and therefore cell viability is unlikely to be devices with NGC-0295 cells are at the borderline of the detection affected. Going forward, strategies to increase stress resistance of level for the NGF immunohistochemistry method, as previously the ECs may still warrant investigation. described.15 Importantly, our study shows a marked improvement The EC biodelivery technology has potential challenges in the in bioactivity when using the NGC0211 cell line for delivery of clinic, including safety of the surgery, the risk for infections at the NGF. The diffusion distance of visible immunoreactive NGF from implantation sites and the recurring need for device replacement. the implants with NGC0211 cells was up to 2 mm. While outside At this point, a safe and tolerable implantation of EC Biodelivery the striatal target area, biologically relevant NGF doses may be devices with NGC-0295 cells has been performed in AD patients. present at even farther distances from the devices, an important No infections at the implantation sites occurred and the notion for the clinical setting. subsequent explantations after 12 months were easily performed Though our primary indication for the NGF-releasing EC and revealed intact devices with no tissue adherence. Importantly, Biodelivery devices is AD, we have tested the bioactivity in the a relevant improvement in cognition and biomarkers in some of excitotoxic striatal QA model, which is modelling many aspects of the AD patients was shown (Eriksdotter-Jo¨nhagen et al., submitted Huntington’s disease. However, QA is also likely to have a role in manuscript). We have further boosted the NGF expression in the pathogenesis of other neurodegenerative diseases, including our human ARPE-19 platform cell line using the SB transposon AD.29,30 Furthermore, the striatum is a sufficiently large structure expression technology, and the current study validates that the to accommodate the device and it contains cholinergic inter- resulting NGC0211 cell line shows improved performance and neurons, responsive to NGF. In accordance with earlier studies provides a promising advance for future clinical devices. Safety delivering NGF by virus or ECs in the QA lesion model,23,24 we aspects and efficacy of clinically sized devices housing found a protective effect of the NGF-secreting devices. The NGC0211 cells are therefore currently being tested in a large protective effect was not as potent as for other neurotrophic animal model with the future goal of performing a NGF dose factors, such as CNTF or meteorin.26 However, our main scope escalation in EC Biodelivery treatment of the cognitive decline was to validate the NGF doses delivered by the two cell lines in in AD patients. a neurodegenerative model. The NGF dose delivered from NGC-0295 was sufficient to significantly improve performance in functional tests. However, the improved performance of NGC0211 cells was illustrated by the small, but significantly MATERIALS AND METHODS higher, improvement in the NGC0211 group in the placement test Establishment of the NGF-expressing NGC0211 cell line at the last testing point, the positive effect on animal growth rate The NGF expression vector pCIn.hNGF was cloned as previously and the observation that only the group with NGC0211 cells had a described.15 From pCIn.hNGF, pCAn.hNGF was cloned by replacing the general lesion size significantly smaller than the group with BglII/BamHI-CI promoter fragment with the corresponding cytomegalovirus parental cells. early enhancer element/chicken beta-actin (CA) promoter sequence from In the normal rat brain, the NGF release from explanted devices pCAIB, a kind gift from Dr E Arenas, Karolinska Institute, Sweden. The with both cell lines increased, compared with pre-implantation contiguous hNGF and neomycin resistance expression cassettes were values. The high release of NGF from the explanted membrane excised as one fragment from pCAn.hNGF with BglII and SspI. This without cells indicates that this increase may to some extent be fragment was inserted between the BglII and EcoRV sites in the SB caused by release of NGF accumulated in the devices in vivo substrate vector pT2BH to create the pT2.CAn.hNGF plasmid. The ARPE-19 (in the unstirred fluid of the membrane and interior device cell line16 was cultured and transfected as previously described.15 Cells components). In addition, the change from the brain environment were co-transfected with plasmids pT2.CAn.hNGF and pCMV-SB-100X with into culture medium may possibly affect the NGF expression from the hyperactive version of the SB transposase (both the SB substrate vector cells in the devices. Though the post-explant release testing does pT2BH and the hyperactive SB transposase were kind gifts from not necessarily reflect the amount of NGF released in vivo, it yields Drs Zsuzsanna Izsvak and Zoltan Ivics, MDC, Berlin, Germany). The plasmid important additional information if the cell line is viable and does not contain a eukaryotic selection marker cassette and is, thus, functioning. NGF secretion of devices in the naive rat was intentionally only transiently expressed. The transient expression window maintained throughout the implantation period. However, in the allows for the active, transposase-mediated integration of the SB QA model, long-term NGF secretion from NGC0211 cells seemed transposon, that is, the inverted repeat SB substrate sequences and the compromised. The nuclei quantifications did not indicate less sequences contained within these repeats. Clones were selected using surviving NGC0211 cells, compared with NGC-0295 cells. However, G418 (Sigma-Aldrich, Copenhagen, Denmark) and single colonies were the QA injections apparently affect the NGF release of the cells isolated and expanded. Clones producing high levels of NGF were further negatively, either by influencing cell health and/or metabolism. In characterised, and NGC0211 was chosen from its ability to deliver long- accordance with this, a previous study with ECs in this model also term NGF expression in conventional cell culture and during encapsulation showed decreased NGF release after device explantation.24 In cell in vitro and in vivo. A stable long-term NGF production was determined culture, the survival and NGF release of both cell clones were not in NGC0211 cells, cultured continuously under standard cell culture

& 2012 Macmillan Publishers Limited Gene Therapy (2012) 1010 --1017 Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1016 conditions (passaged twice a week) for 8.5 months (at least 85 passages) In vivo potency of devices in normal striatum with regular NGF release measurements (1--2 times a month). Before surgery, rats (n ¼ 12) were anaesthetised with isofluran (3--4%) and positioned in a stereotaxic frame (Stoelting, Dublin, Ireland). A midline Characterisation of secreted NGF incision was made in the scalp and two bilateral holes drilled through the Conditioned media samples from cells were processed for western blotting scull. Devices filled with NGC0211 (n ¼ 8), NGC-0295 (n ¼ 8) or ARPE-19 36 cells (n 8) were bilaterally implanted in striatum by an implantation as previously described, and NGF was detected using a goat anti-human ¼ NGF antibody (R&D Systems, Arbingdon, UK). The western blots were run cannula mounted to the stereotaxic frame. The implantation coordinates ± in triplicates and a representative blot is shown. The percentage of mature with respect to Bregma were: AP:0.0, ML: 3.2, DV:À7.5 and TB:À3.3. After implantations, the skin was suture closed. After 2 weeks, rats were deeply and partly processed NGF was quanitified using ImageJ 1.44 software anaesthetised, decapitated and the brains removed. The devices were (Bethesda, MD, USA). 1 For mass spectrometry, cell culture medium harvested from the retrieved and incubated at 37 C in HE-SFM. Media samples for NGC0211 cell line was acidified to pH 4 using 5 M HCl and then determination of NGF release were collected the next day. The brains were immersion fixed in formalin solution for 48 h before cryoprotection in centrifuged for 30 min at 10 000 g. The supernatant was filtered through a 0.45 mixed cellulose filter (HAWP, Millipore, Copenhagen, Denmark), 30% (W/V) sucrose solution in 0.1 M sodium phosphate-buffered saline. In an additional experiment for determination of tissue levels, rats (n 6) applied to a C4 HPLC column, 4.6 Â 250 mm, and eluted with a gradient ¼ from solvent A (0.1% trifluoroacetic acid in H O) to solvent B (0.1% were bilaterally implanted as described above with devices filled with 2 NGC0211 and NGC-0295 cells, respectively. After 3 weeks, rats were killed trifluoroacetic acid in acetonitrile): 10--60% B in 50 min. Fractions (1 min) were collected and assayed by NGF ELISA as described below. The NGF- and the brains removed. Tissue punches were taken in a radius of 2 mm immunoreactive fractions were purified in two further steps using around the implant site and immediately frozen in dry ice. Reference punches were taken in cortex and cerebellum. Samples were homogenised respectively a phenyl and a C8 column, both 2.1 Â 150 mm, and both as previously described,15 and NGF ELISA was performed on diluted eluted with a 15--35% B gradient in 40 min; fractions of 0.5 min were collected. All HPLC columns were from Vydac (Hesperia, CA, USA) and were samples. Explanted devices were placed directly in HE-SFM, either intact or mounted in a 1090 HPLC (Hewlett-Packard, Waldbronn, Germany). Mass with the membrane separated from the cell-containing yarn fraction. After 24 h, NGF release in the media samples was determined by NGF ELISA. spectrometric analyses were performed using an Autoflex II Tof/Tof instrument (Bruker, Bremen, Germany). The molecular mass was obtained Devices, manufactured at the same time, but kept in vitro during the using a-cyano-4-hydroxy-cinnamic acid as matrix, while 1.5-diamino- experimental period were included as references. NGF release from the ± naphtalene was used for top--down sequencing using in-source-decay.37 membranes is expressed as the mean STDEV of that from intact devices. After taking the media samples for NGF measurements, intact devices as well as the membranes without cell-containing yarn were minced with a Encapsulation of cells in EC Biodelivery devices scalpel and sonicated. DNA contents were determined by addition of The 7-mm-long polyether sulphone membranes (Akzo Membrana, Hoechst H 33258 (Invitrogen, Tåstrup, Denmark) and measurement in a Wuppertal, Germany) were fitted with polyvinylalcohol cylindrical foam DQ300 Hoefer fluorometer. No DNA was detected in the membrane, (Clinicel, M-PACT, Eudora, KS, USA). A load tube was attached to the confirming the absence of cells. membrane in one end with ultraviolet-cured acrylic-based glue, and the other end was sealed with the same glue. Before filling, cells were cultured In vivo potency of devices in the rat QA lesion model in growth medium. Cells were dissociated and suspended at a density of Devices (n ¼ 8 in each group) were unilaterally implanted in the right À1 10,000 cells ml in human endothelial serum-free medium (HE-SFM) striatum as described above. Two weeks after device implantations, rats (Invitrogen, Copenhagen, Denmark). A volume of 5 ml of cell solution (n ¼ 32) received an intrastriatal QA injection of 225 nmol as previously 4 (5 Â 10 cells in total) was injected into each device. Devices were kept in described26 with the coordinates adjusted to: TB, À3.3, AP: 1.0; ML: 2.6 and HE-SFM at 37 1C and 5% CO2 for 2--3 weeks before implantations. Devices DV: À5.0 with respect to Bregma. Animals were tested for striatal without cells were treated in the same manner and included as negative dysfunction before QA lesion and 2 and 4 weeks after using the cylinder controls in some experiments. and forelimb placing test, as also previously described.26 All behavioural analyses were performed in a blinded fashion. After the last behavioural Device analyses test 4 weeks after the lesion, the animals were euthanised and immediately The amount of NGF released by each capsule over a 4-h incubation period perfused with cold isotonic saline through the left ventricle. The brains and in HE-SFM was measured using NGF sandwich ELISA (R&D Systems). devices were processed as described above. Standards and samples were assayed in duplicate according to manu- facturer’s instructions, and results were expressed in ng NGF per 24 h. Immunohistochemistry Devices were subsequently fixed in 4% formalin solution, dehydrated in Frozen brains were cut in six series of 40 mm on a freezing microtome. Free- graded ethanol series and embedded in historesin (Leica Microsystems, floating sections were quenched of endogenous peroxidase activity

Ballerup, Denmark). Sections (5 mm) were mounted on poly-L-lysine-coated through incubation with 3% H2O2. Sections were then incubated overnight slides and stained with haematoxylin--eosin (Bie & Berntsen, Rødovre, with goat anti human NGF (R&D Systems), mouse anti-ChAT (Chemicon, Denmark). Images of sections were prepared by a Hamamatsu Copenhagen, Denmark), goat anti-mouse p75NGFR (R&D Systems) or Nanozoomer 1.0-HT scanner (Ballerup, Denmark). mouse anti-NeuN (Chemicon) antibodies diluted in potassium phosphate- buffered saline containing 2% normal horse serum and 0.25% Triton X-100 Animals followed by incubation with the appropriate (anti-mouse or anti-goat) biotinylated secondary antibody. This was followed by conjugation of The experiments involving animals were conducted in accordance with the horseradish peroxidase using a streptavidin--horseradish peroxidase Danish Animal Protection Law and the American Association for Accreditation of Laboratory Animal Care and with experimental procedures complex (ABC elite kit, Vector Laboratories, Petersborough, UK), incubation approved by the Danish National Committee for Ethics in Animal Research with DAB and precipitation of the chromophore with 1% H2O2. Labelled sections were slide mounted and cover slipped. Whole brain images were or the Institutional Animal care and Use Committee (US), respectively. Female Sprague-Dawley (MolTac: s.d., Taconic, Lille Skensved, Denmark) prepared using a Hamamatsu Nanozoomer 1.0-HT scanner. weighing around 220 g at the start of the experiment were used for the study in normal rats. Male Sprague-Dawley (Harlan, Somerville, NJ, USA) Image analyses weighing 250--300 g were used in the QA lesion study. Animals were Visiomorph software (VisioPharm, Hørsholm, Denmark) was used to housed 2--3 per cage with free access to food and water in a standardised analyse images. The mean NGF-IR area was quantiﬁed in serial sections 12-h light/dark cycle. covering the implant. Data are expressed as mean area per section

Gene Therapy (2012) 1010 --1017 & 2012 Macmillan Publishers Limited Transposon-mediated gene transfer for encapsulated cell biodelivery L Fjord-Larsen et al 1017 (mm2)±s.e.m., n ¼ 8 implants. The number and size of striatal ChAT-IR 12 Emerich DF, Winn SR, Harper J, Hammang JP, Baetge EE, Kordower JH. Implants of neurons was determined in a radius of 2 mm from the implant in serial polymer-encapsulated human NGF-secreting cells in the nonhuman primate: sections covering the device implant sites. The number is expressed as rescue and sprouting of degenerating cholinergic basal forebrain neurons. neurons per section ±s.e.m. and the size as mm2 per neuron±s.e.m. J Comp Neurol 1994; 349: 148 --164. 13 Lindner MD, Kearns CE, Winn SR, Frydel B, Emerich DF. Effects of intraventricular (n 6 --8 sections per implant and 8 implants per group)). The area of ¼ encapsulated hNGF-secreting fibroblasts in aged rats. Cell Transplant 1996; 5:205--223. p75NGFR-IR fibres was determined in serial sections covering the device 14 Winn SR, Hammang JP, Emerich DF, Lee A, Palmiter RD, Baetge EE. Polymer- 2 implant site and is expressed as the mean area per section (mm )±s.e.m. encapsulated cells genetically modified to secrete human nerve growth factor (n ¼ 6 --8 sections per implant and 8 implants per group). Quantification of promote the survival of axotomized septal cholinergic neurons. Proc Natl Acad Sci lesion area was performed on NeuN-stained sections. This was done at USA 1994; 91: 2324 --2328. sections þ 1.5, þ 0.5, À0.5 and À1.5 from Bregma, where the lesioned 15 Fjord-Larsen L, Kusk P, Tornoe J, Juliusson B, Torp M, Bjarkam CR et al. Long-term area was manually indicated. To estimate the relative number of living cells delivery of nerve growth factor by encapsulated cell biodelivery in the Gottingen in device sections, the area of nuclei was quantified on 20 Â objective minipig basal forebrain. Mol Ther 2010; 18: 2164 --2172. images from scanned sections, covering the entire device (two sections per 16 Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996; 62: 155 --169. device). The researcher performing the analysis was blinded to the identity 17 Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of sleeping of the experimental groups. beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997; 91: 501 --510. Statistics 18 Ivics Z, Izsvak Z. The expanding universe of transposon technologies for gene and SigmaPlot (Systat, Chicago, IL, USA) was used to analyse the data. For each cell engineering. Mob DNA 2010; 1:25. data set, groups were compared using one-way analysis of variance followed 19 Hui EK, Wang PC, Lo SJ. Strategies for cloning unknown cellular flanking DNA by all pairwise multiple comparison procedures (Dunn’s method or Holm-- sequences from foreign integrants. Cell Mol Life Sci 1998; 54: 1403 --1411. 20 Klein RL, Hirko AC, Meyers CA, Grimes JR, Muzyczka N, Meyer EM. NGF gene Sidaks method) with P-values 0.05 indicating statistical significance. o transfer to intrinsic basal forebrain neurons increases cholinergic cell size and protects from age-related, spatial memory deficits in middle-aged rats. Brain Res CONFLICT OF INTEREST 2000; 875: 144 --151. 21 Bishop KM, Hofer EK, Mehta A, Ramirez A, Sun L, Tuszynski M et al. Therapeutic LF-L, PK, JT, MT and LUW are employed by NsGene A/S. This study was funded by potential of CERE-110 (AAV2-NGF): targeted, stable, and sustained NGF delivery NsGene A/S. and trophic activity on rodent basal forebrain cholinergic neurons. Exp Neurol 2008; 211: 574 --584. 22 Kordower JH, Chen EY, Mufson EJ, Winn SR, Emerich DF. Intrastriatal implants of ACKNOWLEDGEMENTS polymer encapsulated cells genetically modified to secrete human nerve growth We wish to thank Dr Zsuzsanna Izsva´k and Dr Zoltań Ivics for introducing us to the factor: trophic effects upon cholinergic and noncholinergic striatal neurons. sleeping beauty transposon system as well as the generous assistance with NGF copy Neuroscience 1996; 72:63--77. number determination. We thank Helle Nymark, Janni Larsen, Juliano Olsen, 23 Frim DM, Uhler TA, Short MP, Ezzedine ZD, Klagsbrun M, Breakefield XO et al. Marianne Kureer, Pia Knudsen, Hanne Fosmark and Helle T Wassmann for excellent Effects of biologically delivered NGF, BDNF and bFGF on striatal excitotoxic technical assistance with cells and devices. Also, the technical assistance by Allan lesions. Neuroreport 1993; 4: 367 --370. Kastrup with NGF purification and mass spectrometry analysis is highly appreciated. 24 Emerich DF, Hammang JP, Baetge EE, Winn SR. Implantation of polymer- encapsulated human nerve growth factor-secreting fibroblasts attenuates the behavioral and neuropathological consequences of quinolinic acid injections into REFERENCES rodent striatum. Exp Neurol 1994; 130: 141 --150. 1 Jonhagen ME. Nerve growth factor treatment in dementia. Alzheimer Dis Assoc 25 Emerich DF, Mooney DJ, Storrie H, Babu RS, Kordower JH. Injectable hydrogels Disord 2000; 14 (Suppl 1): S31 --S38. providing sustained delivery of vascular endothelial growth factor are neuropro- 2 Rosenberg MB, Friedmann T, Robertson RC, Tuszynski M, Wolff JA, Breakefield tective in a rat model of Huntington’s disease. Neurotox Res 2010; 17:66--74. XO et al. Grafting genetically modified cells to the damaged brain: restorative 26 Jørgensen JR, Emerich DF, Thanos C, Thompson LH, Torp M, Bintz B et al. Lentiviral effects of NGF expression. 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