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

基本信息

批准号：
10467042
负责人：
ANASTASIA KHVOROVA
金额：
$ 137.05万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
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/10467042
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

项目摘要

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.

项目概要工程化 CRISPR 系统有潜力通过以下方式改变遗传性疾病的治疗：尽管如此，基于基因组编辑的治疗仍然是安全、有效且针对目标组织的 CRISPR 递送。效应蛋白及其小RNA引导是临床应用的主要障碍。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药物（例如 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取得的成功：(i) 完整化学修饰（即去除或修饰 100% 2’-OH 基团）的重要性无需纳米颗粒配方即可赋予代谢稳定性并抑制免疫系统激活；使用附加的化学共轭物来调节生物分布特性并接合细胞表面成分这些原则应该能够安全、有效地提供 CRISPR 指南，无论是预加载到其蛋白质效应器中[核糖核蛋白 (RNP) 递送] 或与 mRNA 串联施用或在后一种情况下，编码效应蛋白的病毒载体从载体中解偶联引导RNA递送。基于效应器的传递有望带来额外的好处，包括：（1）改进的引导-目标复用（在并行或串联），（2）在所需的时间后通过目标组织中的自我靶向来灵活地清除病毒基因组编辑已发生，或来自辅助组织（以限制诱导脱靶的长期效应子表达） (3) 通过更精确地聚焦组织特异性编辑的能力引导和效应的正交靶向部分，以及（4）释放其他载体基因组的能力尽管完全修饰、缀合、自我传递的 CRISPR 指导 RNA 具有临床前景，但它们该提案的目标是建立和优化此类指导RNA作为新的指导RNA。 CRISPR 基因组编辑的治疗方式，与效应蛋白递送的多种途径相结合。我们已经确定了一个框架，可以对大多数情况下的指导 RNA 进行完全修饰和稳定。我们还开发了常用的 CRISPR 效应器 (SpyCas9)。提高 DNA 供体的效力和稳定性，从而指导精确修复，这是许多疾病所需的。建议将我们的核酸修饰框架与我们已建立的靶向名册相结合，疏水性、内体溶解性和药代动力学修饰缀合物，使安全有效将基因组编辑机器递送至体内中枢神经系统、肌肉和肾脏组织，首先在小鼠中，然后在猪中，我们将使用 SpyCas9 和其他三个编辑效应器来实现这一目标。此外，我们将建立与我们共同提供修复模板的能力。修改了 RNP 和病毒共传递格式的指南，以实现体内成功的精确基因修复。完成拟议的工作将实现治疗性基因组编辑的重要的新交付能力。