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

Viviana Gradinaru，加州理工学院 随着CRISPR/Cas9等技术的出现， 基础研究和治疗应用正在成为现实。一个杰出的挑战 是安全有效地将大基因组转移到所需细胞的手段 跨度。我们已经开发了一个体内基于Cre-based选择平台（CREATE）， 识别腺相关病毒（AAV），其有效地在遗传学上定义了 人口。我们使用CREATE来选择病毒， 血管内输送，并发现了一种载体，非特异性转导大多数细胞跨越 成人大脑由于血脑屏障的限制性本质， 我们的发现有可能使治疗CNS疾病的障碍 通过CRISPR-Cas或RNA干扰进行基因编辑/替换的令人兴奋的进展， 如果能够达到所需的效率和特异性水平， 专为患病目标设计的 我们计划通过创建基于病毒的非侵入性整体解决方案来实现这些努力， 通过以下方式从胚胎到成人跨越血脑屏障的脑大货物递送： 1.产生用于跨血脑的细胞类型和区域特异性基因递送的AAV- 通过成年啮齿动物的血流非侵入性地屏障神经退行性疾病 应用. 2.产生能够转导子宫内发育中的大脑的AAV， 全身注射给妊娠母鼠用于神经发育研究和治疗。 3.将AAV的包装能力提高约2倍，以实现大规模 基因组用于基因治疗和研究。 4.通过使用系统性的， 载体和遗传编码的活性调节剂（例如通过化学遗传学或其他方法， 现在的发展）。 从长远来看，我们计划在我们的实验室和合作者中， 神经技术（包括，除了病毒载体，组织清除和光遗传学 控制和成像）来阐明适应不良的神经回路， 神经变性和神经发育的脑病理学。

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.