Gene Therapy And Neurodegeneration

基因治疗和神经退行性疾病

基本信息

批准号：
7733798
负责人：
Brandon Harvey
金额：
$ 49.77万
依托单位：
NATIONAL INSTITUTE ON DRUG ABUSE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7733798
关键词：
Affect Astrocytes BMP7 gene Biological Assay Bone Morphogenetic Proteins Brain Brain-Derived Neurotrophic Factor Cell Survival Cells Collaborations Corpus striatum structure Cultured Cells Data Denervation Dopamine Drug toxicity Embryo Frameshift Mutation GDNF gene Gene Delivery Gene Proteins Genes Green Fluorescent Proteins Hypoxia Illicit Drugs Impairment In Vitro Infection Laboratories MPTP Poisoning Methamphetamine Modeling Mus Nerve Degeneration Neuraxis Neurobiology Neuroglia Neurons Parkinson Disease Play Property Protein Overexpression Rattus Reagent Recombinants Rodent Role Serotyping Signal Transduction Pathway Stroke Testing Tissues Toxic effect Transgenes Tropism Viral adeno-associated viral vector cell type day excitotoxicity gene therapy immunoreactivity in vitro Model in vivo interest neurotrophic factor novel prevent protein expression therapeutic gene

项目摘要

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 cell viability 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). As part of a collaboration on a primary project of Dr. Carl Lupica, we found that AAV-GDNF delivered to mouse striatum prior to MPTP lesioning significantly protected against the loss of dopamine and prevented the blockade of corticostriatal LTP. These data demonstrate that dopamine plays a role in supporting several forms of striatal plasticity and that GDNF expression via AAV prevents the loss of dopamine and striatal plasticity caused by MPTP. We propose that impairment of striatal plasticity after dopamine denervation plays a role in the symptomology of Parkinson's disease and that AAV expression of neurotrophic factors represents a tenable approach to protecting against or slowing these neurobiological deficits. We will continue to use AAV vectors for in vivo gene delivery to the brain in models of stroke, Parkinsons disease and methamphetamine-toxicity. Specifically, we evaluate the protective and regenerative properties of a two novel neurotrophic factors Mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopaminergic neurotrophic factor (CDNF). Currently, we are testing the role of AAVMANF and AAVCDNF in models of stroke, Parkinsons disease and methamphetamine-toxicity.

重组腺相关病毒（RAAV）载体经常用于基因传递到中枢神经系统，并能够在体外传递神经元和神经胶质。我们表征了七种表达绿色荧光蛋白（GFP）的RAAV载体的血清型，用于源自胚胎大鼠脑的原代皮质细胞的毒性和毒性。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，但在转盘后6天，除了AAV5以外，递交后表达是神经元的。 AAV2和9产生的最小GFP表达。使用细胞活力测定，除了AAV2和9以外，所有血清型在较高的多重感染（MOI）下观察到毒性。AAV1和5-8的毒性主要影响神经胶质，这表明是由于缺失了神经胶质标记的免疫反应性。 GFP基因中的移码突变降低了血清型1、5和6的总体毒性，但没有7和8，这表明毒性不仅是由于GFP的过表达。总的来说，在具有总体优先神经胶质转导和毒性的原代皮质培养物的AAV血清型中观察到了差异性向量和毒性。现在，我们正在使用这些感染参数在甲基苯丙胺毒性，兴奋性，缺氧和帕金森氏病的体外模型中输送电位治疗基因，并表征涉及骨骼形成蛋白（BMP）保护性和再生性质的信号转导途径。作为Carl Lupica博士主要项目的合作的一部分，我们发现在MPTP病变之前，AAV-GDNF在MPTP病变之前递送至小鼠纹状体，从而受到明显保护的，以防止多巴胺的丧失，并阻止了皮质纹状体LTP的阻塞。这些数据表明，多巴胺在支持几种形式的纹状体可塑性中起作用，并且通过AAV的GDNF表达可防止多巴胺和MPTP引起的纹状体可塑性的丧失。我们认为，多巴胺神经膜治疗后纹状体可塑性的损害在帕金森氏病的症状中起作用，而神经营养因子的AAV表达代表了保护或减慢这些神经生物学缺陷的一种长期方法。我们将继续使用AAV矢量在中风，帕金森氏病和甲基苯丙胺毒性的模型中向体内基因传递到大脑。具体而言，我们评估了两个新型神经营养因子中脑脑胶质细胞衍生的神经营养因子（MANF）和保守的多巴胺能神经营养因子（CDNF）的保护性和再生性质。目前，我们正在测试AAVMANF和AAVCDNF在中风，帕金森氏病和甲基苯丙胺毒性模型中的作用。