Intrabody-dependent activation of cell-specific gene expression in CNS

 描述(申请人提供)：哺乳动物中枢神经系统(CNS)包含数百种分子和功能不同的细胞类型，它们构成神经电路的基本构件。可以使用转基因和转基因动物来标记和操纵单个细胞类型，但这种方法速度慢、成本高，而且范围有限。此外，它不能应用于高等灵长类动物或人类。我们建议开发一种方法，允许从一系列哺乳动物物种中选择性地靶向野生型个体的单个中枢神经系统细胞类型。这是最近开发的一种名为Cre-Dog的技术的改进，该技术使用成对的骆驼纳米体在GFP存在的情况下构建功能性分裂Cre重组酶的组装。我们建议使用这种通用的方法，利用基于Fn3的重组单体来靶向内源性细胞亚型特异的转录因子，这种重组单体可以在体外快速生产和筛选，并用这些单体诱导分裂的Cre和Dre重组酶的组装。这些试剂可以用来诱导通过电穿孔或病毒载体传递的报告和效应器构建体表达的细胞特异性激活。作为这一方法的原理证明，我们将首先使用Fn3-Basd抗GFP单体对在体内构建分裂的Cre和Dre的组装。接下来，我们将提出一对针对细胞特异性视网膜转录因子的单体，并证明 它们可以为功能性Cre重组酶的组装搭建支架，并开发允许这些试剂依赖于Cre的表达的表达结构，以避免单体表达的潜在干扰效应。在此之后，我们将证明这些试剂直接在新生儿视网膜中激活细胞特异性Cre。最后，如果被证明成功，我们将产生一系列试剂，使小鼠和人类的视网膜和大脑皮层主要细胞类型中的报告和效应器结构能够选择性地激活。

 DESCRIPTION (provided by applicant): The mammalian central nervous system (CNS) contains many hundreds of molecularly and functionally distinct cell types, which comprise the basic building blocks of neural circuitry. Individual cell types can be labeled and manipulated using transgenic and knock-in animals, but this approach, is slow, expensive, and limited in scope. Furthermore, it cannot be applied to higher primates or humans. We propose to develop an approach that will allow the selective targeting of individual CNS cell types in wildtype individuals, from a range of mammalian species. This is a modification of a recently developed technology known as CRE-DOG that uses pairs of camelid nanobdies to scaffold assembly of functional split Cre recombinase in the presence of GFP. We propose to use this general approach to target endogenous cell subtype-specific transcription factors using Fn3-based recombinant monobodies, which can be rapidly produced and screened in vitro, and use these to induce assembly of split Cre and Dre recombinase. These reagents can then be used to induce cell-specific activation of expression of reporter and effector constructs delivered by electroporation or viral vector. As proof of principle for this approach, we will first use Fn3-basd pairs of anti- GFP monobodies to scaffold assembly of split Cre and Dre in vivo. We will next raise pairs of monobodies against cell-specific retinal transcription factors, and demonstrate that these can scaffold assembly of functional Cre recombinase, and develop expression constructs that allow Cre-dependent expression of these reagents to avoid potential disruptive effects of monobody expression. Following this, we will demonstrate that these reagents direct cell-specific Cre activation in neonatal retina. Finally, if proven successful, we will generate a toolbx of reagents that will enable selective activation of reporter and effector constructs in the major cell types of retina and cerebral cortex, in both mice and humans.