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

项目摘要 工程化的CRISPR系统有可能通过以下方式改变遗传性疾病的治疗： 基因组编辑疗法尽管如此，CRISPR的安全、有效和靶组织特异性递送仍然是一个挑战。 效应蛋白和它们的小RNA向导是临床应用的主要障碍。因为 小RNA指导的核心重要性，CRISPR的临床开发可以受益于技术 为早期的核酸治疗剂如siRNA和反义寡核苷酸而开发。 两个关键的认识导致了这些治疗方式最近成功的激增： 完全化学改性的重要性（即，除去或修饰100%的2 ′-OH基团）， 在没有纳米颗粒制剂的情况下赋予代谢稳定性并抑制免疫系统活化;和（ii）所述纳米颗粒制剂 附加的化学缀合物用于调节生物分布特性和接合细胞表面组分的用途 促进吸收。这些原则应该能够安全，有效地提供CRISPR指南，无论是在 装载到它们的蛋白质效应物中[核糖核蛋白（RNP）递送]或与mRNA串联施用，或 编码效应蛋白的病毒载体。在后一种情况下，将引导RNA递送从载体解偶联。 基于效应物的递送承诺了额外的益处，包括：（1）改进的引导-靶多路复用（在 并行或串联），（2）在所需的靶向后，通过在靶组织中的自靶向来清除病毒基因组的灵活性， 编辑已经发生，或来自辅助组织（以限制诱导脱靶的延长的效应子表达）。 编辑和免疫反应），（3）通过以下方式更精确地聚焦组织特异性编辑的能力： 用于引导物和效应物的正交靶向部分，和（4）释放载体基因组用于其它靶向部分的能力。 目的尽管完全修饰的、缀合的、自我递送的CRISPR指导RNA的临床前景， 仍然不发达。该提案的目标是建立和优化这样的指导RNA作为新的 CRISPR基因组编辑中的治疗模式，结合多种效应蛋白递送途径。 我们已经确定了一个框架，完全修饰和稳定的指导RNA的最 通常部署的CRISPR效应子（SpyCas 9）。我们还开发了化学修饰， 提高DNA供体的效力和稳定性，指导许多疾病所需的精确修复。我们 建议联合收割机我们的核酸修饰框架与我们建立的靶向， 疏水的、内体溶解的和药代动力学修饰的缀合物，以使得能够安全和有效地 将基因组编辑机器递送到体内中枢神经系统、肌肉和肾脏的组织， 首先是在老鼠身上，然后是在猪身上。我们将使用SpyCas 9和其他三个编辑效应器来实现这一目标， 互补属性。此外，我们还将建立与我们的 RNP和病毒共递送形式的修饰的指导物，以实现体内精确的基因修复。成功 拟议工作的完成将实现治疗性基因组编辑的重要的新递送能力。

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.