Engineering Efficient and Controllable Base Editors

工程高效且可控的碱基编辑器

• 批准号: 10209723
• 负责人: Rahul Manu Kohli
• 金额: $ 43.77万
• 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/10209723
• 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; 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.

这项提议的目的是操纵DNA脱氨酶来产生高度活性和可控的碱基 可以作为精确基因编辑目标的编辑人员。AID对免疫球蛋白基因座的碱基编辑， AID/APOBEC胞嘧啶脱氨酶家族的祖先成员，通常启动 B细胞中的抗体反应，而APOBECs提供对逆转录病毒的保护。在他们的生理上 背景，当DNA脱氨酶由催化受损的CRIPSR/Cas蛋白指导时，它们的碱基编辑 活性可用于在所需的基因组位置引入靶向突变。虽然该系统提供了 出于生物或治疗目的编辑基因组的潜在强大手段，碱基编辑有两种 限制其在基础研究和翻译研究中更广泛应用的障碍。首先，DNA脱氨酶 自然进化为受限制的酶，总体催化活性较低，因为过度激活与 致癌基因突变增加。其次，当调控失调时，AID/APOBEC已知在 他们的目标是促进癌症突变、染色体易位和对化疗的耐药性。 鉴于DNA脱氨酶的自然调控约束在碱基编辑复合体中丢失，这些结构 对基因组构成类似的风险。在这项提议中，我们利用了我们对该机制的广泛知识， 脱氨酶的结构和功能，以克服这些挑战。其一，过度活跃 已经产生了脱氨酶来克服自然减弱的活性，我们将利用这些变体 评估增加脱氨率可以提高基础编辑效率的假设 反应，同时提高精度。其次，我们已经设计出了只能被 在一个小分子的控制下，在靶点上重组。这一战略新提供了 时空控制，这是一项关键要求，将有助于在实验室中使用基本编辑程序，并且是必不可少的 应用于患者的治疗。考虑到基本编辑器的广泛潜在用途，我们将 展示跨不同基因组位置的效率和控制的重要性，然后具体 通过产生表达增强嵌合抗原受体的T(CAR-T)细胞作为模型系统。这些工具 在这里开发的将作为基础编辑在全球范围内推进脱氨酶，并将很容易转化为其他创新 在CRISPR/CAS蛋白中，以及更广泛的基因组工程中。