Circuit-Specific Delivery of Large Cargo Across the Nervous Systems of Adult Mammals and Embryos via Novel Engineered Systemic Vectors

维维亚纳·格拉迪纳鲁，加州理工 随着CRISPR/Cas9等技术的出现，对这两种生物的基因组工程 基础研究和治疗应用正在成为现实。一个突出的挑战 是一种将大基因组安全有效地转移到所需细胞的手段 跨度。我们已经开发了一个基于体内CRE的选择平台(Create)，用于 识别有效转导基因定义的腺相关病毒(AAV) 人口。我们使用CREATE来选择病毒，这些病毒在 血管内传递，并发现了一种非特异性地转导大多数细胞的载体 成人的大脑。因为血脑屏障的限制性是一种主要的 治疗中枢神经系统疾病的障碍我们的发现有可能使 通过CRISPR-CAS或RNA干扰进行基因编辑/替换的令人兴奋的进展 如果可以达到所需的效率和特异度水平，则可以恢复患病的中枢神经系统回路 为患病的目标设计的。 我们计划通过创建基于病毒的解决方案来实现这些努力，以实现非侵入性的整体- 脑大件货物通过血脑屏障从胚胎运送到成人： 1.生成AAVs，用于通过血脑传递细胞类型和区域特定的基因- 成年啮齿动物神经退行性变的非侵入性屏障 申请。 2.用简单的AAVs生成能够在子宫内传导发育中的大脑的AAVs 用于神经发育研究和治疗的怀孕母体全身注射。 3.将自动对讲机的包装能力提高约2倍，以便能够运送大型 用于基因治疗和研究的基因组。 4.通过使用全身性脑深部调制实现非侵入性电路特定的脑深部调制 载体和基因编码的活性调节子(例如，通过化学遗传学或其他 现在发展)。 从长远来看，我们计划在我们的实验室以及与合作者一起，为我们的 神经技术(除病毒载体外，还包括组织清除和光遗传 控制和成像)，以阐明导致 神经退行性变和神经发育中的脑病理学。

Viviana Gradinaru, Caltech With the advent of technologies such as CRISPR/Cas9, genome engineering for both basic research and therapeutic applications is becoming reality. An outstanding challenge is the mean to safely and efficiently transfer large genomes to desired cells across life span. We have developed an in vivo Cre-based selection platform (CREATE) for identifying adeno-associated viruses (AAVs) that efficiently transduce genetically defined populations. We used CREATE to select for viruses that transduce the brain after intravascular delivery and found a vector that nonspecifically transduces most cells across the adult brain. Since the restrictive nature of the blood brain barrier presents a major impediment toward treating CNS disorders our discovery has the potential to enable exciting advances in gene editing/replacement via CRISPR-Cas or RNA interference to restore diseased CNS circuits if the needed level of efficiency and specificity can be engineered for diseased targets. We plan to enable such efforts by creating viral-based solutions to non-invasive whole- brain large cargo delivery across the blood-brain barrier from embryo to adult by: 1. Generating AAVs for cell-type and region specific gene delivery across the blood-brain- barrier, noninvasively via the bloodstream in the adult rodent for neurodegeneration applications. 2. Generate AAVs capable of transducing the developing brain in utero with a simple systemic injection to the pregnant dam for neurodevelopment research and therapy. 3. Increase the packaging capability of AAVs by about 2-fold to enable delivery of large genomes for gene therapy and research. 4. Enable non-invasive circuit specific deep brain modulation by the use of systemic vectors and genetically encoded activity modulators (e.g. by chemogenetics or others in development now). Longer term we plan, in our laboratory and also with collaborators, to contribute our neurotechnologies (including, in addition to viral vectors, tissue clearing and optogenetic control and imaging) towards elucidating maladaptive neural circuits that contribute to brain pathology in neurodegeneration and neurodevelopment.