Gene Therapy And Neuroprotection

重组腺相关病毒（rAV）载体经常用于向中枢神经系统进行基因递送，并且能够在体外转导神经元和神经胶质。 我们的特点是七个血清型的rAAV载体表达绿色荧光蛋白（GFP）的向性和毒性的原代皮质细胞来自胚胎大鼠脑。 在转导后2天，血清型1、5、6、7和8主要在神经胶质中表达GFP，但在转导后6天，除了AAV 5外，表达是神经元的。AAV 2和9产生最小GFP表达。使用LDH和MTS测定，对于除AAV 2和9之外的所有血清型，在较高的感染复数（MOI）下观察到毒性。 AAV 1和5-8的毒性主要影响神经胶质，如神经胶质标志物免疫反应性的丧失所示。 GFP基因中的移码突变降低了血清型1、5和6的总体毒性，但不降低血清型7和8的总体毒性，这表明毒性不仅仅是由于GFP的过表达。 总的来说，在原代皮质培养物上的AAV血清型之间观察到不同的向性和毒性，具有总体优先的胶质细胞转导和毒性。 我们现在正在使用这些感染参数，以提供潜在的治疗基因在体外模型的甲基苯丙胺毒性，兴奋性毒性，缺氧和帕金森氏病，以及表征的信号转导通路参与骨形态发生蛋白（BMP）的保护和再生性能。 我们的第二种方法使用AAV载体在中风、帕金森病和甲基苯丙胺毒性模型中体内递送至大脑。具体而言，我们评估了BMP 7及其信号分子的保护和再生特性。 目前，我们正在测试AAVBMP 7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.