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

通过对引导体和供体进行化学修饰来增强体细胞组织中的 CRISPR 基因编辑

• 批准号: 10671171
• 负责人: ANASTASIA KHVOROVA
• 金额: $ 33.38万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10671171
• 关键词: Animal Model; Animal Testing; Animals; Antisense Oligonucleotides; Area; Biodistribution; C57BL/6 Mouse; Categories; Cell surface; Chemicals; Chemistry; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Maintenance; DNA Modification Process; DNA Repair Pathway; DNA cassette; Development; Digestion; Disease; Drug Kinetics; ENG gene; Engineering; Evaluation; Excision; Family suidae; Fluorescence; Formulation; Generations; Genes; Genome; Genomics; Goals; Guide RNA; Hereditary Disease; Hydrophobicity; Immune response; Immune system; Immunization; Institutes; Kidney; Mediating; Messenger RNA; Metabolic; Methods; Modality; Modification; Mus; Muscle; Neuraxis; Nucleic Acids; Oligonucleotides; Orthologous Gene; Pathway interactions; Phase; Property; Proteins; RNA; Reporter; Reporting; Research; Ribonucleoproteins; Route; Sequence Homology; Series; Small Interfering RNA; Small RNA; Somatic Cell; Specificity; System; Technology; Therapeutic; Tissues; Variant; Viral; Viral Genome; Viral Vector; Work; Writing; base; base editing; biological research; chemical conjugate; clinical application; clinical development; clinical efficacy; clinical practice; delivery vehicle; flexibility; gene repair; gene therapy; genome editing; improved; in vivo; in vivo evaluation; manufacturing scale-up; medical schools; meetings; nanoparticle; novel therapeutics; nuclease; nucleic acid-based therapeutics; prevent; programs; repaired; risk minimization; success; therapeutic RNA; therapeutic genome editing; uptake; vector

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.

项目摘要 工程CRISPR系统有可能通过通过 基于基因组编辑的治疗方法。尽管如此，安全，有效和目标组织特定的CRISPR 效应蛋白及其小的RNA指南代表了临床应用的主要障碍。因为 小型RNA指南的至关重要，CRISPR的临床开发可能会受益于技术 开发了早期的核酸疗法，例如siRNA和反义寡核苷酸。 两种关键的认识导致了这些治疗方式最近取得了成功的激增：（i） 完全化学修饰的重要性（即，2'-OH组的100％去除或修饰） 会议代谢稳定性并抑制没有纳米颗粒配方的免疫系统激活； （ii） 附加的化学共轭物的实用性调整生物分布特性并参与细胞表面组件 这有助于吸收。这些原则应使CRISPR指南的安全有效地提供，要么 加载到其蛋白质作用[核糖核蛋白（RNP）递送]中或与mRNA或 编码效应蛋白的病毒载体。在后一种情况下，解偶联引导从矢量传递RNA- 基于效应子的交付承诺其他好处，包括：（1）改进的指南目标多路复用（在 平行或串联），（2）在所需的目标组织中通过自动靶向清除病毒基因组的灵活性 进行了编辑或来自辅助组织（以限制影响不靶向的效应子表达 （3）通过 正交针对指南和效应子的靶向部分，以及（4）载体基因组能力的解放 目的。尽管临床有望得到完全修改，共轭，自我保留的CRISPR指南RNA，但他们还是 保持欠发达。该提案的目的是建立和优化此类指南RNA作为新的 CRISPR基因组编辑中的治疗方式，以及多种效应蛋白递送的途径。 我们已经确定了一个框架，以最大程度地修改和稳定导向RNA 通常部署的CRISPR效应子（SpyCAS9）。我们还开发了化学修饰 根据许多疾病的需要，提高直接维修精度维修的DNA供体的效力和稳定性。我们 提议将我们的核酸修饰框架与我们已建立的目标， 疏水，内溶胶和药代动力学修改偶联物以实现安全有效 将基因组编辑机械传递到中枢神经系统，肌肉和肾脏的组织，体内 首先在小鼠，然后是猪。我们将使用spycas9实现这一目标，并具有其他三种编辑效果 完全属性。此外，我们还将建立与我们的合并维修模板的能力 RNP和病毒共传递格式的修改指南，以实现体内精确的基因修复。成功的 拟议工作的完成将实现热基因组编辑的重要新输送能力。