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Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secreted protein which reduces endoplasmic reticulum (ER) stress and has neurotrophic effects on dopaminergic neurons. Intracortical delivery of recombinant MANF protein protects tissue from ischemic brain injury in vivo. In this study, we examined the protective effect of adeno-associated virus serotype 7 encoding MANF in a rodent model of stroke. An AAV vector containing human MANF cDNA (AAV-MANF) was constructed and verified for expression of MANF protein. AAV-MANF or an AAV control vector was administered into three sites in the cerebral cortex of adult rats. One week after the vector injections, the right middle cerebral artery (MCA) was ligated for 60 min. Behavioral monitoring was conducted using body asymmetry analysis, neurological testing, and locomotor activity. Standard immunohistochemical and western blotting procedures were conducted to study MANF expression. Our data showed that AAV-induced MANF expression is redistributed in neurons and glia in cerebral cortex after ischemia. Pretreatment with AAV-MANF reduced the volume of cerebral infarction and facilitated behavioral recovery in stroke rats. In conclusion, our data suggest that intracortical delivery of AAV-MANF increases MANF protein production and reduces ischemic brain injury. Ischemia also caused redistribution of AAV-mediated MANF protein suggesting an injury-induced release.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100β, but not the neuronal markers NeuN, MAP2, or β-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15–20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.

Viral vector toxicity is a primary concern in choosing an optimal gene delivery vehicle for either studying biological phenomena or evaluating potential therapeutic strategies. Generally, adenoassociated viral vectors are thought to be among the least toxic viral vectors. Currently, over 100 serotypes of AAV have been identified or produced and the tropism of the serotypes varies. In the central nervous system, studies have shown serotypes 1, 2, 5, 6, 7, 8, 9 and 10 are capable of transduction. We have previously reported the transduction of primary neuronal cultures by serotypes 1, 2, 5, and 6. Here, we compare the toxicity of serotypes 1, 2, 5, 6, 7, 8 and 9 and tropism of serotypes 7, 8 and 9 on primary cortical cultures derived from rat embryonic brain tissue. A self-complementing AAV vector containing modified AAV2 terminal repeats flanking a green fluorescent protein expressed from the CMV promoter was packaged using the cap gene from AAV serotypes 1, 2, 5, 6, 7, 8, or 9. All vectors were purified by CsCl centrifugation. Cultures were infected on DIV6 then examined for GFP fluorescence, toxicity and immunoreactivity on DIV8 and 12. At 48 hours after infection at a comparable MOI of 10000, only serotype 1 showed significant toxicity as determined by LDH assay. Using an MTS assay, similar toxicity was observed at an MOI of 100000; however, at a higher MOI, serotypes 1, 6, 7 and 8 exhibited greater toxicity than serotypes 2, 5 and 9. One week after infection, serotypes 7 and 8 resulted in primarily NeuN+ colocalized with GFP fluorescence, however, GFAP+ fibers expressing GFP were detected. Serotype 9 infected both NeuN+ and GFAP+ cells. Overall, AAV serotypes 1, 2, 5, 6, 7, 8 and 9 transduce rat neurons and glia in vitro with varied efficiency and toxicity.

One prominently used viral vector, adeno-associated virus (AAV), successfully transduces cells of the central nervous system in vivo. Studies have compared the in vivo transduction efficiency of several AAV serotypes, primarily 1, 2 and 5. Here, we compare the transduction of cells in primary neuronal cultures derived from rat embryonic brain tissue using the AAV serotypes 1, 2, 5 and 6, purified by CsCI centrifugation. A self-complementing AAV vector containing AAV2 terminal repeats flanking a green fluorescent protein expressed from the CMV promoter was packaged using the cap gene from AAV serotypes 1, 2, 5 or 6. Cultures were treated on DIV6 then examined by immunofluorescence 7 days later. Compared to untreated cultures, all four serotypes did not alter cell viability as measured by cell permeability assays (LDH release and nuclear dye exclusion). At comparable MOIs, serotypes 1 and 6 yielded a higher number of GFP+ cells than types 2 and 5. At an MOI of 20,000, serotypes 1, 2 and 6 expressed GFP in NeuN+ or GFAP+ cells with similar efficiency. At the same MOI, serotype 5 expressed GFP in more GFAP+ than NeuN+ cells. However, with all four serotypes tested, approximately 10|[ndash]|20% of the GFP+ cells were negative for both GFAP and NeuN. These results demonstrate that AAV serotypes 1, 2, 5 and 6 can transduce both neurons and astrocytes in vitro with minimal toxicity and exhibit a similar transduction profile to that observed in vivo.