Engineering Efficient and Controllable Base Editors

工程高效且可控的碱基编辑器

• 批准号: 10609857
• 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/10609857
• 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; Rattus; Reaction; Resistance; Retroviridae; Risk; Role; Single-Stranded DNA; 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; chimeric antigen receptor T cells; cost; disease-causing mutation; ds-DNA; frontier; genome editing; genomic locus; improved; innovation; insertion/deletion mutation; insight; member; next generation; precise genome editing; programs; 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.

该提案旨在操纵DNA脱氨酶以产生高度活跃且可控的碱基 编辑器，可以针对精确的基因编辑。通过AID对免疫球蛋白基因座进行碱基编辑， 胞嘧啶脱氨酶AID/APOBEC家族的祖先成员，通常启动 APOBECs可以在B细胞中产生抗体应答，而APOBECs则提供针对逆转录病毒的保护。出于他们的生理原因 在上下文中，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 活性可用于在所需基因组基因座处引入靶向突变。虽然该系统提供了一个 潜在的强大手段来编辑基因组用于生物或治疗目的，碱基编辑器有两个 这些障碍限制了它们在基础研究和转化研究中的广泛应用。首先，DNA脱氨酶具有 自然进化为具有低总体催化活性的受限酶，因为超活化与 致癌突变增加第二，当失调时，已知AID/APOBEC在细胞外起作用。 他们的目标，促进癌症诱变，染色体易位和耐化疗。 鉴于对DNA脱氨酶的天然调控约束在碱基编辑复合物中丢失，这些构建体 对基因组也有类似的风险。在这个建议中，我们利用我们对机制的广泛了解， 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天然减弱的活性，我们将利用这些变体 以评估增加脱氨速率可以提高碱基编辑效率的假设 反应，同时提高精度。第二，我们设计出了一种分裂脱氨酶， 在小分子的控制下，在靶向基因座处重构。该战略新提供 时空控制，一个关键的要求，将促进在实验室中使用基本编辑器，是必不可少的 涉及患者的治疗应用。考虑到基编辑器的广泛潜在用途，我们将 证明效率和控制的重要性，广泛跨越不同的基因组位点，然后具体 通过产生增强的表达嵌合抗原受体的T（CAR-T）细胞作为模型系统。的工具 这里开发的将在全球范围内推进脱氨酶作为基础编辑器，并将很容易转化为其他创新 在CRISPR/Cas蛋白中，以及更广泛地涉及基因组工程。