Enhancing CRISPR Gene Editing in Somatic Tissues by Chemical Modification of Guides and Donors

项目摘要 工程CRISPR系统有可能通过以下方式改变遗传病的治疗 以基因组编辑为基础的治疗。然而，CRISPR的安全、有效和靶向组织特异性递送 效应蛋白和它们的小RNA引导是临床应用的主要障碍。因为 小RNA指南的核心重要性，CRISPR的临床开发可以受益于技术 为前几代核酸疗法，如siRNA和反义寡核苷酸而开发。 两个关键的认识导致了最近这些治疗方式的成功：(I) 完全化学修饰(即100%移除或修饰2‘-OH基团)对 在没有纳米颗粒制剂的情况下提高代谢稳定性和抑制免疫系统的激活；以及(Ii) 添加的化学结合物在调节生物分布特性和结合细胞表面成分方面的应用 这有助于人们的理解。这些原则应能够安全、有效地交付CRISPR指南，无论是在 装入它们的蛋白质效应器[核糖核蛋白(RNP)传递]或与mRNAs或 编码效应蛋白的病毒载体。在后一种情况下，解偶联引导RNA从载体- 基于效应器的传输承诺了其他好处，包括：(1)改进的导向-目标多路传输(in 并行或串联)，(2)通过目标组织中的自靶向在所需的 已发生编辑，或来自辅助组织(以限制导致脱靶的长时间效应器表达 编辑和免疫反应)，(3)通过以下方式更精确地聚焦组织特定编辑的能力 导向和效应器的正交靶向部分，以及(4)释放载体基因组容量 目的。尽管完全修饰的、结合的、自我递送的CRISPR引导RNA在临床上有希望，但它们 仍然不发达。这项建议的目标是建立和优化这样的指南RNA作为一种新的 CRISPR基因组编辑中的治疗方式，结合多种效应器蛋白输送途径。 我们已经确定了一个完整修改和稳定大部分引导RNA的框架 通常部署的CRISPR效应器(SpyCas9)。我们还开发了化学修饰， 提高DNA捐赠者的效力和稳定性，指导许多疾病所需的精确修复。我们 建议将我们的核酸修饰框架与我们已建立的靶向、 疏水性、内溶和药代动力学修饰的结合物，使安全有效 将基因组编辑机器运送到体内的中枢神经系统、肌肉和肾脏组织， 首先在老鼠身上，然后在猪身上。我们将使用SpyCas9和其他三个编辑效应器来实现这一目标 互补属性。此外，我们还将内置与我们的 RNP和病毒共传递形式的改良指南，使体内精确的基因修复成为可能。成功 拟议工作的完成将实现治疗性基因组编辑的重要新交付能力。

Project Summary Engineered CRISPR systems have the potential to transform the treatment of inherited diseases via genome editing-based cures. Nonetheless, safe, effective, and target-tissue-specific delivery of CRISPR effector proteins and their small RNA guides represents a major barrier to clinical application. Because of the central importance of the small RNA guides, CRISPR’s clinical development could benefit from technologies developed for earlier generations of nucleic acid therapeutics such as siRNAs and antisense oligonucleotides. Two critical realizations have led to a surge of recent successes with these therapeutic modalities: (i) the importance of complete chemical modification (i.e., the removal or modification of 100% of 2’-OH groups) to confer metabolic stability and suppress immune system activation without nanoparticle formulation; and (ii) the utility of appended chemical conjugates to tune biodistribution properties and engage cell-surface components that facilitate uptake. These principles should enable the safe, effective delivery of CRISPR guides, either pre- loaded into their protein effectors [ribonucleoprotein (RNP) delivery] or administered in tandem with mRNAs or viral vectors that encode the effector protein. In the latter case, uncoupling guide RNA delivery from vector- based effector delivery promises additional benefits including: (1) improved guide-target multiplexing (in parallel or in series), (2) flexibility to clear viral genomes via self-targeting in the target tissue after the desired editing has occurred, or from ancillary tissues (to limit prolonged effector expression that induces off-target editing and immune responses), (3) the ability to more precisely focus tissue-specific editing through orthogonal targeting moieties for guide and effector, and (4) the liberation of vector genomic capacity for other purposes. Despite the clinical promise of fully modified, conjugated, self-delivering CRISPR guide RNAs, they remain underdeveloped. The goal of this proposal is to establish and optimize such guide RNAs as a new therapeutic modality in CRISPR genome editing, in conjunction with multiple routes of effector protein delivery. We have identified a framework for complete modification and stabilization of guide RNAs for the most commonly deployed CRISPR effector (SpyCas9). We have also developed chemical modifications that increase the potency and stability of DNA donors that direct precise repairs, as needed for many diseases. We propose to combine our nucleic acid modification framework with our established roster of targeted, hydrophobic, endosomolytic, and pharmacokinetics-modifying conjugates to enable the safe and effective delivery of the genome editing machinery to tissues of the central nervous system, muscle and kidney in vivo, first in mice and then in pigs. We will pursue this goal with SpyCas9 and with three other editing effectors with complementary attributes. In addition, we will build in the capability to co-deliver repair templates with our modified guides, in both RNP and viral co-delivery formats, to enable precise gene repairs in vivo. Successful completion of the proposed work will realize important new delivery capabilities for therapeutic genome editing.