Engineered Cas9 Nucleases with Single-Genomic-Site Precision for CYBB Correction

用于 CYBB 校正的具有单基因组位点精度的工程化 Cas9 核酸酶

基本信息

批准号：
9272917
负责人：
ERIK J. SONTHEIMER
金额：
$ 38.88万
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：
2016
资助国家：
美国
起止时间：
2016-05-12 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9272917
关键词：
Ablation Affect Affinity Alpha Cell Attenuated Autologous Biological Assay CD34 gene CRISPR/Cas technology Cell Therapy Cell Transplantation Cells Characteristics Chimera organism Chronic Granulomatous Disease Clinical Complex DNA DNA Binding DNA Binding Domain DNA Repair Defect Development Dimerization Disease Endonuclease I Engineering Ensure Enzymes Event Exhibits Family Future Gene Targeting Genes Genetic Genome Genome engineering Genomics Goals Guide RNA Hematopoietic stem cells Human Genome Immune Inherited Licensing Life Link Location Modification Mutation Myeloid Cells Neisseria meningitidis Orthologous Gene Pathway interactions Patients Phagocytes Pharmaceutical Preparations Process Property Protein Engineering Proteins Reagent Regulation Research Science Site Specificity Staphylococcus aureus Stem cells Streptococcus pyogenes System Technology Therapeutic Transcription Coactivator Treatment Efficacy Variant Viral Zinc Fingers base biological systems cellular engineering clinical application curative treatments design dimer experimental study gene correction gene therapy genome editing genome-wide improved innovation knockout gene nuclease programs prototype public health relevance repaired stem cell therapy technological innovation therapeutic gene tool vertebrate genome

项目摘要

DESCRIPTION (provided by applicant) Type II CRISPR/Cas9 systems are revolutionizing biomedical science. These programmable nucleases facilitate the creation of a double strand break at a specific location within a genome, which promotes targeted gene disruption or gene editing through homologous repair with an exogenously supplied donor DNA. While existing Cas9 systems are powerful, their promiscuity presents a barrier to their implementation in gene therapy applications, where undesired collateral damage to the treated genome must be minimized or, ideally, eliminated. Consequently, further development of this nuclease platform for the selective recognition and cleavage of a desired target sequence (and only that sequence) is warranted. To achieve the ultimate goal of single-site nuclease precision within the human genome, we propose to develop a chimeric fusion between Cas9 and a programmable DNA-binding domain (pDBD). We have established and validated a working prototype that has improved precision, greater activity, and a broader sequence targeting range than the standard Cas9 system. In this proposal, we outline experiments to use appended pDBDs to improve precision of three representative, validated Cas9 orthologs: S. pyogenes and S. aureus Cas9 (SpCas9 & SaCas9; representative Type II-A) and N. meningitidis Cas9 (NmCas9; representative Type II-C). These systems will be applied to the therapeutic gene correction of chronic granulomatous disease (CGD). In Aim 1, we will optimize the characteristics of our established SpCas9-pDBD fusions to create a chimeric system that requires an additional stage of licensing for target cleavage and incorporates exogenous regulation through a drug-dependent dimerization system. In Aim 2, we will extend the advantages of Cas9-pDBD fusions into the more compact NmCas9 and SaCas9, and identify the similarities and differences in essential design principles between Type II-A and Type II-C Cas9-pDBD fusions. In Aim 3, we will apply our Cas9-pDBD system to the precise and efficient correction in hematopoietic stem cells of X-linked defects that are associated with CGD to establish a gene correction-based autologous stem cell therapy for this devastating disease. Ultimately, the proposed research promises to yield genome-editing enzymes that exhibit the specificity required for safe, effective application in clinical gene therapy and stem cell engineering, which we will demonstrate by creating a cell-based gene therapy for CGD.

描述（由申请人提供） II 型 CRISPR/Cas9 系统正在彻底改变生物医学科学，这些可编程核酸酶有助于在基因组内的特定位置产生双链断裂，从而通过与现有的外源供体 DNA 进行同源修复来促进靶向基因破坏或基因编辑。 Cas9系统功能强大，但它们的混杂性对其在基因治疗应用中的实施造成了障碍，在基因治疗应用中，必须尽量减少或理想情况下消除对基因组的不良附带损害，从而进一步发展。为了实现人类基因组内单位点核酸酶精确度的最终目标，我们建议开发 Cas9 和可编程序列之间的嵌合融合。 DNA 结合域 (pDBD)。我们已经建立并验证了一个工作原型，该原型比标准 Cas9 系统具有更高的精度、更高的活性和更广泛的序列靶向范围。在本提案中，我们概述了使用附加的实验。 pDBD 可提高三种代表性、经过验证的 Cas9 直系同源物的精度：化脓性链球菌和金黄色葡萄球菌 Cas9（SpCas9 和 SaCas9；代表性 II-A 型）和脑膜炎奈瑟菌 Cas9（NmCas9；代表性 II-C 型）。应用于慢性肉芽肿病 (CGD) 的治疗基因校正在目标 1 中，我们将优化已建立的 SpCas9-pDBD 融合体的特征。创建一个嵌合系统，该系统需要额外的靶标切割许可阶段，并通过药物依赖性二聚化系统纳入外源调节。在目标 2 中，我们将 Cas9-pDBD 融合的优势扩展到更紧凑的 NmCas9 和 SaCas9，以及确定 II-A 型和 II-C 型 Cas9-pDBD 融合之间基本设计原则的异同。在目标 3 中，我们将 Cas9-pDBD 系统应用于精确有效地纠正与 CGD 相关的 X 连锁缺陷的造血干细胞，为这种毁灭性疾病建立基于基因纠正的自体干细胞疗法，最终，拟议的研究有望产生具有特异性的基因组编辑酶。这是临床基因治疗和干细胞工程中安全、有效应用所必需的，我们将通过创建基于细胞的 CGD 基因疗法来证明这一点。