Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord.

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo.

Recent advances in the therapeutic uses of chondroitinase ABC.

Many studies, using pre-clinical models of SCI, have demonstrated the efficacy of chondroitinase ABC as a treatment for spinal cord injury and this has been confirmed in laboratories worldwide and in several animal models. The aim of this review is report the current state of research in the field and to compare the relative efficacies of these new interventions to improve outcomes in both acute and chronic models of SCI. We also report new methods of chondroitinase delivery and the outcomes of two clinical trials using the enzyme to treat spinal cord injury in dogs and disc herniation in human patients. Recent studies have assessed the outcomes of combining chondroitinase with other strategies known to promote recovery following spinal cord injury and new approaches. Evidence is emerging that one of the most powerful combinations is that of chondroitinase with cell transplants. The particular benefits of each of the different cell types used for these transplant experiments are discussed. Combining chondroitinase with rehabilitation also improves outcomes. Gene therapy is an efficient method of enzyme delivery to the injured spinal cord and circumvents the issue of the enzyme's thermo-instability. Other methods of delivery, such as via nanoparticles or synthetic scaffolds, have shown promise; however, the outcomes from these experiments suggest that these methods of delivery require further optimization to achieve similar levels of efficacy to that obtained by a gene therapy approach. Pre-clinical models have also shown chondroitinase is efficacious in the treatment of other conditions, such as peripheral nerve injury, stroke, coronary reperfusion, Parkinson's disease and certain types of cancer. The wide range of conditions where the benefits of chondroitinase treatment have been demonstrated reflects the complex roles that chondroitin sulphate proteoglycans (its substrate) play in health and disease and warrants the enzyme's further development as a therapy.

The Chemorepulsive Protein Semaphorin 3A and Perineuronal Net-Mediated Plasticity.

During postnatal development, closure of critical periods coincides with the appearance of extracellular matrix structures, called perineuronal nets (PNN), around various neuronal populations throughout the brain. The absence or presence of PNN strongly correlates with neuronal plasticity. It is not clear how PNN regulate plasticity. The repulsive axon guidance proteins Semaphorin (Sema) 3A and Sema3B are also prominently expressed in the postnatal and adult brain. In the neocortex, Sema3A accumulates in the PNN that form around parvalbumin positive inhibitory interneurons during the closure of critical periods. Sema3A interacts with high-affinity with chondroitin sulfate E, a component of PNN. The localization of Sema3A in PNN and its inhibitory effects on developing neurites are intriguing features and may clarify how PNN mediate structural neural plasticity. In the cerebellum, enhanced neuronal plasticity as a result of an enriched environment correlates with reduced Sema3A expression in PNN. Here, we first review the distribution of Sema3A and Sema3B expression in the rat brain and the biochemical interaction of Sema3A with PNN. Subsequently, we review what is known so far about functional correlates of changes in Sema3A expression in PNN. Finally, we propose a model of how Semaphorins in the PNN may influence local connectivity.

Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors.

Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this article, we compared adeno-associated viral (AAV) vectors based on serotypes 1-9 for their capability to transduce cultured primary rat and human SCs and nerve segments. AAV1 is the best serotype to transduce rat SCs, whereas AAV2 and AAV6 performed equally well in human SCs. Transduction of monolayers of cultured rat and human SCs did not accurately predict the transduction efficiency in nerve segments. Rat nerve segments could be genetically modified equally well by a set of four AAV vectors (AAV1, AAV5, AAV7, AAV9), whereas AAV2 was superior in human nerve segments. The current experiments were undertaken as a first step towards future clinical implementation of ex vivo AAV-based gene therapy in surgical nerve repair. The transduction of rat and human SCs and nerve segments by entirely different AAV serotypes, as documented here, highlights one of the challenges of translating gene therapy from experimental animals to human patients.

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve.

Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.

A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons.

Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the cytomegalovirus (CMV) and chicken beta-actin (CAG) promoters in a back-to-back configuration with a shared enhancer, and show efficient expression of two proteins simultaneously in DRG neurons. We demonstrate how this is useful for experiments on axonal regeneration, by co-expressing a gene of interest and an axonal marker. Using a farnesylated form of eGFP, which is actively transported along axons, we show superior long-distance labelling of axons of DRG neurons compared with normal eGFP. Additionally, we have efficiently transduced lumbar DRG neurons by injecting the AAV dual promoter vector into the dorsal intrathecal space, which is a less invasive delivery method. In summary, we have developed an AAV dual promoter vector designed for simultaneous expression of a gene of interest and a fluorescent protein to label long-distance axonal projections, which allows specific quantification of axons from transduced neurons after injury.

Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector.

The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.

Nerve surgery and gene therapy: a neurobiological and clinical perspective.

Despite major microsurgical improvements the clinical outcome of peripheral nerve surgery is still regarded as suboptimal. Over the past decade several innovative techniques have been developed to extend the armamentarium of the nerve surgeon. This review evaluates the potential of gene therapy in the context of peripheral nerve repair. First the main challenges impeding peripheral nerve regeneration are presented. This is followed by a short introduction to gene therapy and an overview of its most important advantages over the classical delivery of therapeutic proteins. Next, this review focuses on the most promising viral vectors capable of targeting the peripheral nervous system and their first application in animal models. In addition, the challenges of translating these experimental results to the clinic, the limitations of current vectors and the further developments needed, are discussed. Finally, four strategies are presented on how gene therapy could help patients that have to undergo reconstructive nerve surgery in the future.

Early passage bone marrow stromal cells express genes involved in nervous system development supporting their relevance for neural repair.

PURPOSE: The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC. METHODS: Fourty four k whole genome rat microarrays were used to study gene expression of cultured BMSC at passage (P)3 and to compare expression profiles between P3 and P14 BMSC. Quantitative PCR was employed to validate the microarray results. RESULTS: P3 BMSC expressed genes involved in neural developmental events such as glial differentiation, neuron proliferation, and neurite formation. They also express genes encoding for growth factors and for proteins involved in growth factor signaling. A total of 6687 genes were co-expressed in P3 and P14 BMSC. Of these co-expressed genes, 3% (202 genes) was differentially expressed with 159 genes higher in P3 BMSC and 43 genes higher in P14 BMSC. The gene expression patterns were independently validated using quantitative PCR. Functional data mining by Gene Ontology (GO)-analysis revealed that 85/159 and 22/43 genes were annotated in the GO database. In P3 BMSC, 53 GO-classes were overrepresented with several involved in organ development, cell proliferation, and neural repair. In P14 BMSC, three GO-classes were overrepresented with one involved in organ development. CONCLUSIONS: Our gene profiling results suggested a decreased plasticity and repair aptitude of long-term cultured BMSC. Our data indicated the use of early passage BMSC for neural repair approaches.

Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons.

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

Neuroinflammation, as defined by activation of local glial cells and production of various inflammatory mediators, is an important feature of many neurological disorders. Expression of pro-inflammatory mediators produced by glial cells in the central nervous system (CNS) is considered to contribute to the neuropathology observed in those diseases. To diminish the production or action of pro-inflammatory mediators, we have used lentiviral (LV) vector-mediated encoding rat interleukin-10 (rIL-10) or rat interleukin-1 receptor antagonist (rIL-1ra) to direct the local, long-term expression of these anti-inflammatory cytokines in the CNS. We have shown that cultured macrophages or astroglia transduced with LV-rIL-10 or LV-rIL-1ra produced far less tumor necrosis factor (TNF)alpha or IL-6, respectively in response to pro-inflammatory stimuli. Moreover, intracerebroventricular (i.c.v.) administration of LV-rIL-10 or LV-rIL-1ra resulted in transduction of glial cells and macrophages and, subsequently reduced TNFalpha, IL-6 and inducible nitric oxide synthase (iNOS) expression in various brain regions induced by inflammatory stimuli, whereas peripheral expression of these mediators remained unaffected. In addition, expression levels of the anti-inflammatory cytokines IL-4 and transforming growth factor-beta were not altered in either brain or pituitary gland. Furthermore, i.c.v. administration of LV-rIL-10 or LV-rIL-1ra given during the remission phase of chronic-relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, improved the clinical outcome of the relapse phase. Thus, local application of LV vectors expressing anti-inflammatory cytokines could be of therapeutic interest to counteract pro-inflammatory processes in the brain without interfering with the peripheral production of inflammatory mediators.

A spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression after lumbar ventral root avulsion and implantation.

Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection.

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP(+) cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

Gene transfer to the spinal cord neural scar with lentiviral vectors: predominant transgene expression in astrocytes but not in meningeal cells.

Viral vector-mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein (LV-GFP) was injected in and around the neural scar 2 weeks after a dorsal column lesion in the rat spinal cord in order to analyze transduction characteristics of the neural scar after 4, 7, and 14 days. GFP expression was found at all points after injection and increased from 4 to 7 days, with no apparent difference observed between 7 and 14 days. The core of the lesion was virtually devoid of GFP signal despite direct vector injections in this area. The colocalization of GFP with specific cell markers (GFAP, vimentin, Raldh2, NeuN, OX-42, ED-1, and NG-2) indicated that the predominant cells transduced in the rim of the lesion were astrocytes, with neurons, microglia, oligodendrocyte precursors, and macrophages transduced to a lesser extent. None of the Raldh2-positive meningeal cells, present in the core of the scar, expressed GFP. In vitro meningeal cells were readily transduced, indicating that in vivo the formation of an extracellular matrix might prevent LV particles from transducing cells in the core of the scar. Because astrocytes are important cellular constituents of the glial scar after CNS injury, transduction of astrocytes with LV vectors encoding neurotrophic factors like BDNF or NT-3 may be used to enhance regeneration of severed axonal tracts through or along boundaries of a CNS lesion.

International spinal research trust research strategy. III: A discussion document.

STUDY DESIGN: Discussion document. OBJECTIVES/METHODS: To review the Research Strategy of the International Spinal Research Trust (ISRT), which identifies key areas of basic and clinical research that are likely to be beneficial in developing potential treatments for spinal cord injury for funding. This strategy is intended to both guide the programme of research towards areas of priority and stimulate discussion of the different avenues of research. This latest document has been developed to take into account the scientific progress in the 6 years since publication of the previous Research Strategy. RESULTS/DISCUSSION: The latest scientific developments in research designed to repair the spinal cord and restore function following injury and how they might impact on spinal cord injury research are highlighted.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function.

To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding for green fluorescent protein or saline. AAV-BDNF- and AAV-NT-3-transduced 293 human kidney cells produced and secreted BDNF or NT-3, respectively, in vitro. The secreted neurotrophins were biologically active; they both promoted outgrowth of sensory neurites in vitro. In vivo, transgene expression was observed predominantly in neurons for at least 16 weeks after injection. Compared with controls, a modest though significant improvement in hind-limb function was found in rats that received AAV-BDNF and AAV-NT-3. Retrograde tracing demonstrated that twice as many neurons with processes extending toward the Schwann cell graft were present in the second lumbar cord segment of AAV-BDNF- and AAV-NT-3-injected animals compared with controls. We found no evidence, however, for growth of regenerated axons from the Schwann cell implant into the caudal cord. Our results suggest that AAV vector-mediated overexpression of BDNF and NT-3 in the cord caudal to a Schwann cell bridge modified the local lumbar axonal circuitry, which was beneficial for locomotor function.

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord.

Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated viral (rAAV)-mediated over-expression of brain-derived neurotrophic factor (BDNF) in the spinal cord on chronic neuropathic pain after unilateral chronic constriction injury (CCI) of the sciatic nerve. The rAAV-BDNF vector was injected into the dorsal horn at the thirteenth thoracic spinal cord vertebra (L(1) level) 1 week after CCI. Allodynia and hyperalgesia induced by CCI in the hindpaws were permanently reversed, beginning 1 week after vector injection, compared with a similar injection of a control rAAV-GFP vector (green fluorescent protein) or saline. In situ hybridization for BDNF demonstrated that both dorsal and ventral lumbar spinal neurons contained an intense signal for BDNF mRNA, at 1 to 8 weeks after vector injection. There was no similar BDNF mRNA over-expression associated with either injections of saline or rAAV-GFP. These data suggest that chronic neuropathic pain is sensitive to early spinal BDNF levels after partial nerve injury and that rAAV-mediated gene transfer could potentially be used to reverse chronic pain after nervous system injuries in humans.