Development of CNS-targeted AAV vectors

描述 AAV载体通过直接脑实质内输注将基因传递到大脑是非常有效的，在那里它们可能在实验动物的一生中介导持续的转基因表达。现有AAV载体的这些特殊特性是正在进行或计划进行的神经系统疾病临床试验的基础，在这些试验中，局部基因传递在治疗上是有效的。然而，许多其他神经系统疾病的基因治疗方法的发展将需要将全球基因输送到中枢神经系统。在这里，我们将通过体内选择AAV衣壳文库和分子嫁接来开发具有这种功能的新的CNS靶向AAV载体。在第一种方法中，我们将使用体内选择AAV衣壳文库，该文库具有通过DNA改组AAV1、2、5、8、9和RH10帽基因而产生的多样性5x109克隆，以鉴定静脉或ICV注射后的成年动物的新的嗜脑和嗜脊髓的AAV衣壳。该文库将通过成年小鼠的尾静脉或侧脑室注入，一个月后，我们将从大脑和脊髓中提取DNA，用于组织驻留AAVCap基因的PCR扩增。这些克隆将被克隆回原始文库质粒骨架，并用于生产更多的AAV病毒粒子，用于随后几轮体内选择。我们将在每一轮选择中对10个AAV帽基因进行测序。一旦我们确定了每个靶点的组织驻留AAV帽基因的收敛，我们将使用新选择的衣壳制备编码萤火虫荧光素酶(Fluc)的重组AAV载体，并将它们注入成年动物体内，以确定它们的生物分布。我们将使用生物发光成像来评估基因表达的分布和动力学，然后对不同器官中的Fluc活性进行生化定量，并对转导细胞在脑和脊髓中的分布进行组织学评估。在第二种方法中，我们将结合带有不同蛋白质和多肽的AAV载体，这些蛋白质和多肽在血管内输注后可以高效地运送脂质体、酶和siRNAs穿过血脑屏障。为此，我们将使用携带14个氨基酸的生物素受体多肽的嵌合AAV衣壳，允许在包装过程中将生物素合并到AAV衣壳中。作为脑靶向分子，我们将使用链霉亲和素融合到针对小鼠转铁蛋白受体(TFR)的单链抗体，或来自人ApoB-100的多肽，或狂犬病病毒糖蛋白。新的AAV-分子结合物在静脉输注后的生物分布将如上所述进行评估。所有新的针对CNS的AAV载体的治疗效果将在GM1-神经节苷脂沉积症的小鼠模型中进行测试，GM1-神经节苷脂病是一种严重影响CNS的溶酶体储存疾病。我们期望这一新一代以中枢神经系统为靶点的AAV载体能够促进高效基因治疗方法的发展，以治疗目前现代医学无法企及的许多儿童、成人和老年神经疾病。

DESCRIPTION AAV vectors are exceptionally efficient for gene delivery to the brain by direct intraparenchymal infusion, where they mediate continuous transgene expression perhaps for the lifetime of the experimental animal. These exceptional properties of existing AAV vectors are the basis for ongoing or planned clinical trials for neurological diseases where focal gene delivery is therapeutically effective. However development of gene therapy approaches for many other neurological diseases will require global gene delivery to the CNS. Here we will use in vivo selection of an AAV capsid library and molecular grafting to develop new CNS- targeted AAV vectors with such capability. In the first approach we will use in vivo selection of an AAV capsid library with a diversity of 5x109 clones generated by DNA shuffling of AAV1, 2, 5, 8, 9, and rh10 Cap genes, to identify new brain- and spinal cord-tropic AAV capsids after intravenous or ICV delivery in adult animals. The library will be infused via the tail vein or the cerebral lateral ventricles of adult mice and one month later we will isolate DNA from the brain and spinal cord for PCR amplification of tissue-resident AAV Cap genes. These will be cloned back into the original library plasmid backbone and used to produce more AAV virions for subsequent rounds of in vivo selection. We will sequence 10 AAV Cap genes per round of selection. Once we determine convergence of tissue resident AAV Cap genes for each target, we will prepare recombinant AAV vectors encoding firefly luciferase (Fluc) using the newly selected capsids and infuse them into adult animals to determine their biodistribution. We will use bioluminescence imaging to assess distribution and kinetics of gene expression followed by biochemical quantification of Fluc activity in different organs and histological assessment of transduced cell distribution in brain and spinal cord. In the second approach we will combine AAV vectors with different proteins and peptides previously shown to be highly efficient in ferrying liposomes, enzymes, and siRNAs across the BBB after intravascular infusion. For this we will use chimeric AAV capsids carrying a 14 amino acid biotin acceptor peptide that allows for incorporation of biotin into the AAV capsid during packaging. As brain-targeting molecules we will use Streptavidin fused to a single-chain antibody specific for the mouse Transferrin receptor (TfR), or peptides derived from human ApoB-100, or Rabies virus glycoprotein. The biodistribution of the new AAV-molecular conjugates after intravenous infusion will be assessed as above. The therapeutic efficacy of all new CNS-targeted AAV vectors will be tested in a mouse model of GM1-gangliosidosis, which is a lysosomal storage disease that severely affects the CNS. We expect this new generation of CNS-targeted AAV vectors to foster the development of highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine