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

• 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
• 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

 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，从而通过与外源供应的供体 DNA 进行同源修复来促进目标基因破坏或基因编辑。虽然现有的 Cas9 系统功能强大，但其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治疗基因组的不良附带损害。因此，有必要进一步开发该核酸酶平台，以选择性识别和切割所需的靶序列（并且仅该序列）。 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 DNA 结合域 (pDBD) 之间的嵌合融合。我们已经建立并验证了一个工作原型，与标准 Cas9 系统相比，该原型具有更高的精度、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 pDBD 来提高三种代表性、经过验证的 Cas9 直向同源物的精度的实验：化脓性链球菌和金黄色葡萄球菌 Cas9（SpCas9 和 SaCas9；代表性 II-A 型）和脑膜炎奈瑟氏球菌 Cas9（NmCas9；代表性 II-C 型）。这些系统将应用于慢性肉芽肿病（CGD）的治疗性基因校正。在目标 1 中，我们将优化已建立的 SpCas9-pDBD 融合体的特性，以创建一个嵌合系统，该系统需要额外阶段的靶标裂解许可，并通过药物依赖性二聚化系统纳入外源调节。在目标2中，我们将把Cas9-pDBD融合的优势扩展到更紧凑的NmCas9和SaCas9，并确定II-A型和II-C型Cas9-pDBD融合在基本设计原则上的异同。在目标3中，我们将应用Cas9-pDBD系统对与CGD相关的X连锁缺陷的造血干细胞进行精确有效的校正，为这种毁灭性疾病建立基于基因校正的自体干细胞疗法。 最终，拟议的研究有望产生基因组编辑酶，这些酶具有临床基因治疗和干细胞工程安全、有效应用所需的特异性，我们将通过创建基于细胞的 CGD 基因治疗来证明这一点。