Enhancing CRISPR-based Therapeutic Interventions Via a Targeted Platform Tool

通过靶向平台工具加强基于CRISPR的治疗干预 项目摘要 本提案中描述的方法预期了转化药物的需求 通过设计和制造一个广泛适用的平台工具， 基于CRISPR的治疗优先化：Cas9的模块化、靶向、细胞特异性递送。 任何基于细胞的疾病的基因疗法的追求变得更有效，通过该工具的 体外发现阶段的多功能性，体内临床前药物毒性的连续性 阶段，以及人类临床给药的潜力，而不会影响准确性， 基因改变的结果。 本建议书中所述的平台方法包括模块化的灵活性， 链霉亲和素-生物素相互作用提供了一种工具，使药物发现研究人员， 有效地交换出各种抗体，以优先考虑不同的特定细胞靶点，消除 需要为每种感兴趣的细胞类型创建独特的CRISPR工具集。通过利用 使用抗体靶向细胞为基础的疾病，如在癌症干预，目前的建议 打算利用抗体相同的特异性结合特性来递送CRISPR/Cas9 基因相关疗法使研究人员能够在更大的细胞内靶向特定的细胞， 体内或体外异质细胞群预测下游候选药物 验证需求。 该提案的第一阶段描述了一项实验设计，以验证 当掺入Cas9的细胞特异性靶向递送时的CRISPR基因编辑 成分通过在细胞水平上分离靶向递送的基因， 编辑仪器（Cas9）从递送核靶向遗传信息，治疗 这种安全机制增强了潜在的安全性，防止了脱靶基因的改变， 不需要的细胞群体。利用这种“分子手术”技术， 基于受体表达的靶细胞群体，一种通用平台工具，包括 将开发和检查附着于链霉亲和素的Cas9以增强CRISPR- 基于治疗识别。开发过程将包括：1）重组 产生具有用于交联的定制缀合位点的增强的特异性Cas9，2） 包含链霉亲和素和重组Cas9的缀合物的缀合和纯化， 和3）确认Cas9的选择性特异性递送和保留的编辑能力，以删除一个或多个Cas9片段。 干细胞群中的基因。该项目将验证细胞特异性靶向方法 通过体外程序递送Cas9，并以敏锐的眼光测试体内 计划进行第二阶段的测试。

Enhancing CRISPR-based Therapeutic Interventions Via a Targeted Platform Tool Project Summary The approach described in this proposal anticipates the needs of translational drug development by designing and manufacturing a broadly applicable platform tool that eases CRISPR-based therapeutic prioritization: modular, targeted, cell-specific delivery of Cas9. Pursuit of gene therapies for any cell-based disorder becomes more efficient through the tool's versatility during in vitro discovery phases, continuity through in vivo preclinical pharm-tox phases, and potential for human clinical administration without compromising accuracy in gene-altering outcomes. The platform approach described in this proposal incorporates the modular flexibility of streptavidin-biotin interactions to provide a tool that allows drug discovery researchers to efficiently swap-out a variety of antibodies to prioritize different specific cell targets, eliminating the need to create a unique CRISPR toolset for each cell-type of interest. By leveraging the use of antibodies to target cell-based diseases, as in cancer intervention, the current proposal intends to harness the same specific binding properties of antibodies to deliver CRISPR/Cas9 gene-related therapies. Enabling researchers to target specific cells within a larger heterogeneous cell population either in vivo or in vitro anticipates downstream drug candidate validation needs. Phase I of this proposal describes an experimental design to validate the efficiency of CRISPR gene editing when incorporating cell-specific targeted delivery of the Cas9 component. Through separation at the cellular level of the targeted delivery of the gene- editing instrument (Cas9) from the delivery of nucleus-targeted genetic information, therapeutic potential is enhanced by this safety mechanism, preventing off-target gene altering in undesired cell populations. Drawing on this “molecular surgery” technology to specifically target cell-populations based on receptor expression, a universal platform tool comprised of Cas9 attached to Streptavidin will be developed and examined for enhancement of CRISPR- based therapeutic identification. The development process will include: 1) Recombinant production of an enhanced specificity Cas9 with bespoke conjugation sites for crosslinking, 2) Conjugation and purification of a conjugate comprising streptavidin and recombinant Cas9, and 3) Confirmation of selective specific delivery of Cas9 and retained editing ability to delete a gene within stem cell populations. This project will validate the cell-specific targeting approach to deliver Cas9 via in vitro procedures and testing with a keen eye to the needs of in vivo testing planned for Phase II.