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

 描述（由申请人提供）：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，它们构成神经回路的基本构建块。可以使用转基因和敲入动物标记和操纵单个细胞类型，但这种方法缓慢、昂贵且范围有限。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，允许选择性靶向野生型个体中的单个CNS细胞类型，来自一系列哺乳动物物种。这是对最近开发的称为CRE-DOG的技术的修改，该技术使用成对的骆驼纳米体来在GFP存在下支撑功能性分裂Cre重组酶的组装。我们建议使用这种通用的方法来靶向内源性细胞亚型特异性转录因子，使用基于Fn 3的重组单体，其可以在体外快速产生和筛选，并使用这些来诱导分裂Cre和Dre重组酶的组装。然后，这些试剂可用于诱导通过电穿孔或病毒载体递送的报告子和效应子构建体的表达的细胞特异性活化。作为这种方法的原理证明，我们将首先使用基于Fn 3的抗GFP单体对来支撑体内分裂的Cre和Dre的组装。接下来，我们将提出针对细胞特异性视网膜转录因子的单抗体对，并证明， 这些可以为功能性Cre重组酶的组装提供支架，并开发允许这些试剂依赖Cre表达的表达构建体，以避免单体表达的潜在破坏性影响。在此之后，我们将证明这些试剂直接在新生儿视网膜细胞特异性Cre激活。最后，如果被证明是成功的，我们将产生一个试剂的工具箱，将能够选择性激活报告和效应结构的主要细胞类型的视网膜和大脑皮层，在小鼠和人类。

 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.