Gene Therapy And Neuroprotection

重组腺相关病毒（rAAV）载体经常用于中枢神经系统的基因传递，并且能够在体外转导神经元和胶质细胞。我们对表达绿色荧光蛋白（GFP）的rAAV载体的7种血清型在胚胎大鼠脑皮层原代细胞中的趋向性和毒性进行了表征。在转导后2天，血清型1、5、6、7和8主要在胶质细胞中表达GFP，但在转导后6天，除AAV5外，其他血清型均表达GFP。AAV2和aav9的GFP表达量最小。通过LDH和MTS检测，除AAV2和aav9外，所有血清型在较高感染多重度（MOI）时均观察到毒性。AAV1和5-8的毒性主要影响胶质细胞，这表明胶质细胞标记免疫反应性的丧失。GFP基因的移码突变降低了血清型1、5和6的总体毒性，但没有降低血清型7和8的毒性，这表明毒性不仅仅是由于GFP的过表达。总的来说，不同的AAV血清型在原代皮层培养物上观察到不同的趋向性和毒性，总体上具有优先的胶质转导和毒性。我们现在正在使用这些感染参数在甲基苯丙胺毒性、兴奋性毒性、缺氧和帕金森病的体外模型中传递潜在的治疗基因，并表征骨形态发生蛋白（BMPs）保护和再生特性相关的信号转导途径。我们的第二种方法是在中风、帕金森病和甲基苯丙胺毒性模型中使用AAV载体在体内传递到大脑。具体来说，我们评估了BMP7及其信号分子的保护和再生特性。目前，我们正在测试AAVBMP7和AAVSMADs在帕金森病和甲基苯丙胺毒性模型中的作用。

Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. We characterized seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1, 5, 6, 7 and 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using LDH and MTS assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity. We are now using these infection parameters to deliver potentital therapeutic genes in vitro models of methamphetamine-toxicity, excitotoxicity, hypoxia and Parkinsons disease as well as characterize the signal transduction pathways involved in the protective and regenerative properties of bone morphogenetic proteins (BMPs). Our second approach uses AAV vectors for in vivo delivery to the brain in models of stroke, Parkinsons disease and methamphetamine-toxicity. Specifically, we evaluate the protective and regenerative properties of BMP7 and its signaling molecules. Currently, we are testing the role of AAVBMP7 and AAVSMADs in models of Parkinsons and methamphetamine-toxicity.