Unilateral ex vivo gene therapy by GDNF in epileptic rats.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. This neurological disorder is characterized by focal seizures originating in the temporal lobe, often with secondary generalization. A variety of pharmacological treatments exist for patients suffering from focal seizures, but systemically administered drugs offer only symptomatic relief and frequently cause unwanted side effects. Moreover, available drugs are ineffective in one third of the epilepsy patients. Thus, developing more targeted and effective treatment strategies for focal seizures, originating from, e.g., the temporal lobe, is highly warranted. In order to deliver potential anti-epileptic agents directly into the seizure focus we used encapsulated cell biodelivery (ECB), a specific type of ex vivo gene therapy. Specifically, we asked whether unilateral delivery of glial cell line-derived neurotrophic factor (GDNF), exclusively into the epileptic focus, would suppress already established spontaneous recurrent seizures (SRS) in rats. Our results show that GDNF delivered by ECB devices unilaterally into the seizure focus in the hippocampus effectively decreases the number of SRS in epileptic rats. Thus, our study demonstrates that focal unilateral delivery of neurotrophic factors, such as GDNF, using ex vivo gene therapy based on ECB devices could be an effective anti-epileptic strategy providing a bases for the development of a novel, alternative, treatment for focal epilepsies.

Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain.

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

The feasibility of an encapsulated cell approach in an animal deafness model.

For patients with profound hearing loss a cochlear implant (CI) is the only treatment today. The function of a CI depends in part of the function and survival of the remaining spiral ganglion neurons (SGN). It is well known from animal models that inner ear infusion of neurotrophic factors prevents SGN degeneration and maintains electrical responsiveness in deafened animals. The purpose with this study was to investigate the effects of a novel encapsulated cell (EC) device releasing neurotrophic factors in the deafened guinea pig. The results showed that an EC device releasing glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) implanted for four weeks in deafened guinea pigs significantly preserved the SGNs and maintained their electrical responsiveness. There was a significant difference between BDNF and GDNF in favour of GDNF. This study, demonstrating positive structural and functional effects in the deafened inner ear, suggests that an implanted EC device releasing biologically protective substances offers a feasible approach for treating progressive hearing impairment.

A new versatile and compact lentiviral vector.

During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to become highly useful tools for efficient and stable delivery of transgenes to dividing and nondividing cells in a variety of experimental protocols. The vector system has been progressively and substantially improved,mainly to meet growing concerns over safety issues. However, the actual design and size of the lentiviral transfer vector often makes transgene cloning and DNA preparation a troublesome task. In this study, the pHR transfer vector used for lentivirus production in many laboratories was modified to contain a more versatile polylinker than the one present in the original pHR vector. In addition, the vector was significantly reduced in size from 12 to 7 kb, by replacing the original vector backbone with sequence from the multipurpose pUC18 vector. These modifications allowed for easier cloning and higher DNA yields without compromising the fundamental ability of this vector system to transduce cells in vitro and in vivo. Finally, the trimmed vector sequence was fully characterized by sequencing the vector in its entirety. In both cultured cells and directly into the rat striatum, transduction with this lentivirus, based on the modified pHsCXW vector, was as efficient and durable as with the pHR vector-based virus. In conclusion, the modified lentiviral transfer vector pHsCXW holds promise as a new valuable tool for the research community in the field of gene transfer.

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites.

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.

Encapsulated cell therapy for neurodegenerative diseases: from promise to product.

Delivering therapeutic molecules, including trophic factor proteins, across the blood brain barrier to the brain parenchyma to treat chronic neurodegenerative diseases remains one of the great challenges in biology. To be effective, delivery needs to occur in a long-term and stable manner at sufficient quantities directly to the target region in a manner that is selective but yet covers enough of the target site to be efficacious. One promising approach uses cellular implants that produce and deliver therapeutic molecules directly to the brain region of interest. Implanted cells can be precisely positioned into the desired region and can be protected from host immunological attack by encapsulating them and by surrounding them within an immunoisolatory, semipermeable capsule. In this approach, cells are enclosed within a semiporous capsule with a perm selective membrane barrier that admits oxygen and required nutrients and releases bioactive cell secretions while restricting passage of larger cytotoxic agents from the host immune defense system. Recent advances in human cell line development have increased the levels of secreted therapeutic molecules from encapsulated cells, and membrane extrusion techniques have led to the first ever clinical demonstrations of long-term survival and function of encapsulated cells in the brain parenchyma. As such, cell encapsulation is capable of providing a targeted, continuous, de novo synthesized source of very high levels of therapeutic molecules that can be distributed over significant portions of the brain.

Encapsulated cell-based biodelivery of meteorin is neuroprotective in the quinolinic acid rat model of neurodegenerative disease.

PURPOSE: Encapsulated cell (EC) biodelivery is a promising, clinically relevant technology platform to safely target the delivery of therapeutic proteins to the central nervous system. The purpose of this study was to evaluate EC biodelivery of the novel neurotrophic factor, Meteorin, to the striatum of rats and to investigate its neuroprotective effects against quinolinic acid (QA)-induced excitotoxicity. METHODS: Meteorin-producing ARPE-19 cells were loaded into EC biodelivery devices and implanted into the striatum of rats. Two weeks after implantation, QA was injected into the ipsilateral striatum followed by assessment of neurological performance two and four weeks after QA administration. RESULTS: Implant-delivered Meteorin effectively protected against QA-induced toxicity, as manifested by both near-normal neurological performance and reduction of brain cell death. Morphological analysis of the Meteorin-treated brains showed a markedly reduced striatal lesion size. The EC biodelivery devices produced stable or even increasing levels of Meteorin throughout the study over 6 weeks. CONCLUSIONS: Stereotactically implanted EC biodelivery devices releasing Meteorin could offer a feasible strategy in the treatment of neurological diseases with an excitotoxic component such as Huntington's disease. In a broader sense, the EC biodelivery technology is a promising therapeutic protein delivery platform for the treatment of a wide range of diseases of the central nervous system.

Targeted delivery of nerve growth factor via encapsulated cell biodelivery in Alzheimer disease: a technology platform for restorative neurosurgery.

OBJECT: The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants that deliver nerve growth factor (NGF) to the cholinergic basal forebrain with the intention of halting the degeneration of cholinergic neurons and the associated cognitive decline in patients with Alzheimer disease (AD). The NsG0202 implant (NsGene A/S) consists of an NGF-producing, genetically engineered human cell line encapsulated behind a semipermeable hollow fiber membrane that allows the influx of nutrients and the efflux of NGF. The centimeter-long capsule is attached to an inert polymer tether that is used to guide the capsule to the target via stereotactic techniques and is anchored to the skull at the bur hole. METHODS: Six patients with mild to moderate AD were included in this Phase Ib open-label safety study and were divided into 2 dose cohorts. The first cohort of 3 patients received single implants targeting the basal nucleus of Meynert (Ch4 region) bilaterally (2 implants per patient), and after a safety evaluation, a second cohort of 3 patients received bilateral implants (a total of 4 implants per patient) targeting both the Ch4 region and the vertical limb of the diagonal band of Broca (Ch2 region). Stereotactic implantation of the devices was successfully accomplished in all patients. Despite extensive brain atrophy, all targets could be reached without traversing sulci, the insula, or lateral ventricles. RESULTS: Postoperative CT scans allowed visualization of the barium-impregnated tethers, and fusion of the scans with stereotactic MR images scan was used to verify the intended positions of the implants. Follow-up MRI at 3 and 12 months postimplantation showed no evidence of inflammation or device displacement. At 12 months, implants were successfully retrieved, and low but persistent NGF secretion was detected in half of the patients. CONCLUSIONS: With refinement, the ECB technology is positioned to become an important therapeutic platform in restorative neurosurgery and, in combination with other therapeutic factors, may be relevant for the treatment of a variety of neurological disorders. Clinical trial registration no.: NCT01163825.

A surgical device for minimally invasive implantation of experimental deep brain stimulation leads in large research animals.

BACKGROUND: Deep brain stimulation (DBS) in experimental animals has promoted new indications and refined existing treatments. Implantation of downscaled clinical DBS leads directly compatible with commercially available implantable pulse generators can however be challenging. Accordingly, we have developed a lead implantation device (LID) and technique for minimally invasive implantation of experimental multicontact DBS leads in large research animals. MATERIALS AND METHODS: The LID carries a small-diameter biocompatible polyimide guide tube which is first used for inserting the stimulating end of the lead and then implanted subcutaneously with the rest of the lead. The functionality of the device was tested by implanting 2 different designs of experimental DBS leads in 12 Göttingen minipigs for up to 12 weeks. The brains were histologically analyzed in order to assess implantation accuracy and local tissue reaction. RESULTS AND CONCLUSIONS: The LID was easy to handle and capable of accurate stereotaxic implantation of downscaled experimental DBS leads in the predetermined target brain structures with minimal surrounding tissue reaction. The device may benefit future large animal DBS research as it allows for precise implantation of DBS leads and may have implications for further refinement of clinical DBS leads.

Efficient in vivo protection of nigral dopaminergic neurons by lentiviral gene transfer of a modified Neurturin construct.

Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRalpha2 or GFRalpha1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored.

Increased in vitro and in vivo transgene expression levels mediated through cis-acting elements.

BACKGROUND: Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. METHODS: Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. RESULTS: Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. CONCLUSION: The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

Generation and characterization of immortalized rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are involved in, among other things, the pathogenesis of pancreatic fibrosis. Here, we present the generation of immortalized PSCs 7 and 14 days after isolation by retroviral gene transfer of the SV40 large T antigen encoding region. Propagated cell lines [large T immortalized cells (LTC)-7, LTC-14] retained characteristics of primary cells in terms of morphology, responsiveness to mediators regulating cellular functions such as proliferation, and expression profile of a number of investigated genes. Whereas LTC-14 kept the morphological features of the differentiation status of the primary cells they were made of, LTC-7 appeared similar to an earlier stage. Thus the established cell lines represent a versatile tool to investigate various aspects of PSC biology.