Development of CNS-targeted AAV vectors

描述 AAV载体对于通过直接脑实质内输注将基因递送至脑是非常有效的，在脑实质内它们介导连续的转基因表达，可能持续实验动物的一生。现有AAV载体的这些特殊性质是正在进行或计划进行的神经系统疾病临床试验的基础，其中焦点基因递送在治疗上是有效的。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全球基因递送到中枢神经系统。在这里，我们将使用AAV衣壳文库的体内选择和分子移植来开发具有这种能力的新的CNS靶向AAV载体。在第一种方法中，我们将使用AAV衣壳文库的体内选择，该文库具有通过AAV 1、2、5、8、9和rhlO Cap基因的DNA改组产生的5xl 09个克隆的多样性，以在成年动物中静脉内或ICV递送后鉴定新的脑和脊髓嗜性AAV衣壳。文库将通过成年小鼠的尾静脉或大脑侧脑室输注，一个月后，我们将从脑和脊髓分离DNA用于组织驻留AAV Cap基因的PCR扩增。这些将被克隆回原始文库质粒骨架中，并用于产生更多的AAV病毒体用于随后的体内选择轮次。我们将每轮选择测序10个AAV Cap基因。一旦我们确定了每个靶标的组织驻留AAV Cap基因的收敛性，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，并将其注入成年动物中以确定其生物分布。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，并对脑和脊髓中的转导细胞分布进行组织学评估。在第二种方法中，我们将联合收割机AAV载体与不同的蛋白质和肽相结合，这些蛋白质和肽先前被证明在血管内输注后高效地运送脂质体、酶和siRNA穿过BBB。为此，我们将使用携带14个氨基酸生物素受体肽的嵌合AAV衣壳，其允许在包装期间将生物素掺入AAV衣壳中。作为脑靶向分子，我们将使用与小鼠转铁蛋白受体（TfR）特异性单链抗体融合的链霉亲和素，或来自人ApoB-100或狂犬病病毒糖蛋白的肽。将如上评估静脉内输注后新AAV-分子缀合物的生物分布。将在GM 1-神经节苷脂沉积症的小鼠模型中测试所有新的CNS靶向AAV载体的治疗功效，所述神经节苷脂沉积症是一种严重影响CNS的溶酶体贮积病。我们期望这种新一代的CNS靶向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治疗的许多儿童、成人和老年神经系统疾病

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