Engineering Efficient and Controllable Base Editors

工程高效且可控的碱基编辑器

• 批准号: 10396080
• 负责人: Rahul Manu Kohli
• 金额: $ 43.79万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10396080
• 关键词: Address; Adenosine; Antibodies; Antibody Response; Attenuated; B-Lymphocytes; BCAR1 gene; Basic Science; Biochemical; Biological; Biological Models; Biology; Biotechnology; Chromosomal translocation; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cytosine; Cytosine deaminase; DNA; Deaminase; Deamination; Diagnosis; Disease; Distant; Endonuclease I; Engineering; Enzymes; Epigenetic Process; Event; Family; Family member; Generations; Genes; Genetic Engineering; Genome; Genome Stability; Genome engineering; Genomic Instability; Genomics; Guide RNA; Hyperactivity; Immune; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Impairment; Knock-out; Knowledge; Laboratories; Limes; Link; Malignant Neoplasms; Medicine; Molecular; Mutagenesis; Mutate; Mutation; Nature; Nucleic Acids; Nucleosides; Oncogenic; Outcome; Pathway interactions; Patients; Physiological; Play; Positioning Attribute; Property; RNA; RNA Binding; RNA Editing; Rattus; Reaction; Resistance; Retroviridae; Risk; Role; Site; Structure; System; T-Lymphocyte; Terminator Codon; Therapeutic; Transfer RNA; Translating; Translational Research; Uracil; Variant; Work; Zinc; activation-induced cytidine deaminase; anti-cancer; anticancer activity; apoB mRNA editing catalytic subunit; base; base editing; base editor; chemotherapy; chimeric antigen receptor; cost; disease-causing mutation; ds-DNA; frontier; genome editing; genomic locus; improved; innovation; insertion/deletion mutation; insight; member; next generation; precise genome editing; reconstitution; repaired; risk minimization; small molecule; spatiotemporal; tool; transition mutation

This proposal aims to manipulate DNA deaminase enzymes to generate hyperactive and controllable base editors that can be targeted for precise gene editing. Base editing of the immunoglobulin locus by AID, the ancestral member of the AID/APOBEC family of cytosine deaminase enzymes, normally initiates maturation of antibody responses in B-cells, while APOBECs provide protection against retroviruses. Out of their physiological context, when DNA deaminases are directed by catalytically-impaired CRIPSR/Cas proteins, their base editing activity can be used to introduce targeted mutations at a desired genomic locus. While this system offers a potentially powerful means to edit the genome for biological or therapeutic purposes, base editors have two barriers that limit their broader application in basic and translational research. First, DNA deaminases have naturally evolved to be constrained enzymes with low overall catalytic activity, as hyperactivation is associated with increased oncogenic mutations. Second, when dysregulated, AID/APOBECs are known to act outside of their targets, promoting cancer mutagenesis, chromosomal translocations, and resistance to chemotherapy. Given that natural regulatory constraints on DNA deaminases are lost in base editor complexes, these constructs pose similar risks to the genome. In this proposal, we harness our extensive knowledge of the mechanism, structure and function of deaminase enzymes in order to overcome these challenges. For one, hyperactive deaminases have been generated to overcome the naturally attenuated activity, and we will exploit these variants to evaluate the hypothesis that increasing the deamination rate can improve the efficiency of the base editing reaction, while simultaneously improving precision. Second, we have devised split deaminases that can only be reconstituted at the targeted locus under the control of a small molecule. This strategy newly offers spatiotemporal control, a critical requirement that will facilitate the use of base editors in the lab and is essential to therapeutic applications in patients. Given the wide range of potential uses for base editors, we will demonstrate the importance of efficiency and control broadly across diverse genomic sites, and then specifically by generating enhanced chimeric antigen receptor expressing T (CAR-T) cells as a model system. The tools developed here will globally advance deaminases as base editors and will readily translate to other innovations in CRISPR/Cas proteins, and to genome engineering more generally.

该建议旨在操纵DNA脱氨酶以产生过度活跃和可控的碱基 可以针对精确基因编辑的编辑。通过辅助 胞质脱氨酶的援助/APOBEC家族的祖先成员，通常启动成熟 B细胞中的抗体反应，而APOBEC可以保护逆转录病毒。从生理学出来 上下文，当DNA脱氨酶是由催化受损的CRIPSR/CAS蛋白引导的，它们的碱基编辑 活性可用于在所需的基因组基因座上引入靶向突变。虽然该系统提供了 为生物学或治疗目的编辑基因组的潜在强大方法，基础编辑有两个 限制其在基本和转化研究中更广泛应用的障碍。首先，DNA脱氨酶具有 自然进化为受约束的酶，总体催化活性低，因为过度激活与 随着致癌突变的增加。其次，当失调时，已知辅助/apobec在以外行动 它们的靶标，促进癌症诱变，染色体易位和对化学疗法的抗性。 鉴于在基本编辑器复合物中丢失了对DNA脱氨酶的自然调节限制，因此这些构造 与基因组构成类似的风险。在此提案中，我们利用我们对机制的广泛了解， 为了克服这些挑战，脱氨酶的结构和功能。一个，多动 已经生成了脱氨酶来克服自然减弱的活性，我们将利用这些变体 为了评估提高脱氨酸速率可以提高基本编辑效率的假设 反应，同时提高精度。其次，我们设计了只能是 在小分子的控制下在目标基因座进行重构。这个策略新提出 时空控制，这是一项关键要求，可以促进实验室中使用基本编辑器的使用，这是必不可少的 用于患者的治疗应用。鉴于基本编辑器的各种潜在用途，我们将 展示效率和控制的重要性，并在各种基因组部位上广泛进行，然后特别是 通过生成增强的嵌合抗原受体，以模型系统表达T（CAR-T）细胞。工具 这里开发的将在全球范围内作为基础编辑，并很容易转化为其他创新 在CRISPR/CAS蛋白中，更广泛地进行基因组工程。