Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in the rat spinal cord following injury.

Adeno-associated viral (AAV) vectors are a promising system for transgene delivery into the central nervous system (CNS) based on their safety profile and long-term gene expression. Gene delivery to the CNS has largely been neuron centric but advances in AAV technology are facilitating the development of approaches to enable transduction of glial cells. Considering the role of astrocytes in the on-going secondary damage in spinal cord injury (SCI), an AAV vector that targets astrocytes could show benefit as a potential treatment. Transduction efficiency, transgene expression and cellular tropism were compared for the AAV serotypes AAV5, AAV9 and AAVRec2 whereby destabilised yellow fluorescent protein (dYFP) was controlled by the GFAP or the truncated GfaABC1D promoter. The vectors were tested in primary spinal cord astrocyte cell culture, spinal cord slice culture and an in vivo model of SCI contusion. AAV5 resulted in greater transduction efficiency, transgene expression and astrocyte tropism compared with AAV9 and AAVRec2. In a rodent model of SCI, robust transgene expression by AAV5-GFAP/GfaABC1D-dYFP was observed through 12 mm of spinal cord tissue and expression was largely restricted to astrocytes. Thus, AAV5-GFAP/GfaABC1D carries the potential as a potential gene therapy vector, particularly for transducing astrocytes in the damaged spinal cord.

Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

Chondroitin sulphate proteoglycans (CSPGs) are inhibitors to axon regeneration and plasticity. A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) is a human enzyme that catalyses the proteolysis of CSPG protein cores. Infusion of ADAMTS4 into the damaged spinal cord was previously shown to improve functional recovery SCI, however, this therapy is limited in its enzyme form. Adeno-associated viral (AAV) vector gene therapy has emerged as the vector of choice for safe, robust and long-term transgene expression in the central nervous system. Here, an AAV expression cassette containing ADAMTS4 under the control of the astrocytic GfaABC1D promoter was packaged into an AAV5 vector. Sustained expression of ADAMTS4 was achieved in vitro and in vivo leading to degradation of CSPGs. Compared to a contusion only group, AAV-ADAMTS4 resulted in significantly decreased lesion size, increased sprouting of hindlimb corticospinal tract axons, increased serotonergic fiber density caudal to a contusive spinal cord injury. Hindlimb-specific exercise rehabilitation was used to drive neuroplasticity towards improving functional connections. The combination of hindlimb rehabilitation with AAV-ADAMTS4 led to functional recovery after SCI compared to a contusion only group. Thus, long-term degradation of CSPGs through AAV-ADAMTS4 gene therapy in a combinational approach with rehabilitation represents a candidate for further preclinical development.

Connexin43 Expression and Associated Chronic Inflammation Presages the Development of Cerebral Radiation Necrosis.

Cerebral radiation necrosis (CRN) is a delayed complication of radiosurgery that can result in severe neurological deficits. The biological changes leading to necrotic damage may identify therapeutic targets for this complication. Connexin43 expression associated with chronic inflammation may presage the development of CRN. A mouse model of delayed CRN was used. The left hemispheres of adult female mice were irradiated with single-fraction, high-dose radiation using a Leksell Gamma Knife. The brains were collected 1 and 4 days, and 1-3 weeks after the radiation. The expression of connexin43, interleukin-1ß (IL-1ß), GFAP, isolectin B-4, and fibrinogen was evaluated using immunohistochemical staining and image analysis. Compared with the baseline, the area of connexin43 and IL-1ß staining was increased in ipsilateral hemispheres 4 days after radiation. Over the following 3 weeks, the density of connexin43 gradually increased in parallel with progressive increases in GFAP, isolectin B-4, and fibrinogen labeling. The overexpression of connexin43 in parallel with IL-1ß spread into the affected brain regions first. Further intensified upregulation of connexin43 was associated with escalated astrocytosis, microgliosis, and blood-brain barrier breach. Connexin43-mediated inflammation may underlie radiation necrosis and further investigation of connexin43 hemichannel blockage is merited for the treatment of CRN.

PSA-NCAM positive neural progenitors stably expressing BDNF promote functional recovery in a mouse model of spinal cord injury.

BACKGROUND: Neural stem cells for the treatment of spinal cord injury (SCI) are of particular interest for future therapeutic use. However, until now, stem cell therapies are often limited due to the inhibitory environment following the injury. Therefore, in this study, we aimed at testing a combinatorial approach with BDNF (brain-derived neurotrophic factor) overexpressing early neural progenitors derived from mouse embryonic stem cells. BDNF is a neurotrophin, which both facilitates neural differentiation of stem cells and favors regeneration of damaged axons. METHODS: Mouse embryonic stem cells, modified to stably express BDNF-GFP, were differentiated into PSA-NCAM positive progenitors, which were enriched, and SSEA1 depleted by a sequential procedure of magnetic-activated and fluorescence-activated cell sorting. Purified cells were injected into the lesion core seven days after contusion injury of the spinal cord in mice, and the Basso mouse scale (BMS) test to evaluate motor function was performed for 5 weeks after transplantation. To analyze axonal regeneration the anterograde tracer biotinylated dextran amine was injected into the sensorimotor cortex two weeks prior to tissue analysis. Cellular differentiation was analyzed by immunohistochemistry of spinal cord sections. RESULTS: Motor function was significantly improved in animals obtaining transplanted BDNF-GFP-overexpressing cells as compared to GFP-expressing cells and vehicle controls. Stem cell differentiation in vivo revealed an increase of neuronal and oligodendrocytic lineage differentiation by BDNF as evaluated by immunohistochemistry of the neuronal marker MAP2 (microtubule associated protein 2) and the oligodendrocytic markers ASPA (aspartoacylase) and Olig2 (oligodendrocyte transcription factor 2). Furthermore, axonal tracing showed a significant increase of biotin dextran amine positive corticospinal tract fibers in BDNF-GFP-cell transplanted animals caudally to the lesion site. CONCLUSIONS: The combinatorial therapy approach by transplanting BDNF-overexpressing neural progenitors improved motor function in a mouse contusion model of SCI. Histologically, we observed enhanced neuronal and oligodendrocytic differentiation of progenitors as well as enhanced axonal regeneration.