A large-capacity bacterial platform for the production and targeted delivery of gene editing systems

总结 存在对赋予基因编辑技术的广泛治疗用途的递送系统的未满足的需求。 CRISPR/Cas系统的强大功能已被用于各种基因编辑方法， 被誉为治疗性基因编辑的未来。治疗实施的一个关键障碍是 CRISPR/Cas介导的基因编辑策略是所需组分的有效递送，即，两非 将编码RNA和大的Cas9核酸酶结合到靶细胞和组织中。当前的交付平台，例如，病毒 和非病毒平台，具有几个弱点，包括（1）缺乏靶向特异性，2）不能 进入细胞，3）免疫激活，4）脱靶效应和有限的治疗窗口，以及5）有限的遗传 编码和货物容量。SiVEC生物技术公司，在第一阶段和第二阶段SBIR赠款的支持下， NIAID（1 R43 AI 140243 - 01 A1和2 R44 AI 140243 -02）先前已开发出基于细菌的递送 该平台可以有效地产生短发夹RNA（shRNAs）并将其递送到特异性靶向组织。这 平台不限于shRNA的交付，在这个建议中，我们将工程师它创建“SiCRISP”-一个新的 该平台生产并提供CRISPR/Cas9指导的基因编辑所需的所有组件。我们还将 进行概念验证研究，用于将基因编辑机器递送到临床相关组织。 通过应用SiVEC交付平台的关键组件，SiCRISP克服了几个关键的 限制现有基因编辑递送系统的治疗潜力的缺点：（1）它可以靶向特定的 细胞和组织，并且可以通过多种途径施用，例如，鼻内、全身、靶向注射， 眼内、阴道内等;（2）它可以进入细胞并逃离内体以有效地递送其分子。 货物;（3）它已被实验证实为非免疫原性，它不被宿主免疫识别， 细胞，即使在重复递送后;（4）其机制不依赖于宿主基因组整合，因此其作用 是短暂的，减少脱靶效应，限制毒性，提高安全性;（5）对编码没有限制 和输送能力（即，它在一个剂量中同时表达和递送所有CRISPR/Cas9组分）; (6)它是廉价的快速和大量生产;（7）作为一个一体化的系统，它消除了额外的 制造步骤;（8）它可以生产和递送任何基因编辑系统的组分（例如，TALEN， 碱基编辑器和大范围核酸酶），这是由于其编码多功能性和大编码容量。这些创新 SiCRISP递送平台的功能将有助于实现基因编辑治疗癌症的全部潜力。 人类疾病

SUMMARY There is an unmet need for delivery systems that empower broad therapeutic use of gene editing technologies. The power of the CRISPR/Cas system has been harnessed for a variety of gene editing approaches, and it has been hailed as the future of therapeutic gene editing. A key impediment to the therapeutic implementation of CRISPR/Cas-mediated gene editing strategies is the efficient delivery of the required components, i.e., two non- coding RNAs and the large Cas9 nuclease, to target cells and tissues. Current delivery platforms, e.g., viruses and non-viral platforms, suffer from several weaknesses, including (1) lack of targeting specificity, 2) inability to enter cells, 3) immune activation, 4) off-target effects and limited therapeutic window, and 5) limited genetic encoding and cargo capacity. SiVEC Biotechnologies, with support from phase I and phase II SBIR grants from NIAID (1R43AI140243-01A1 and 2R44AI140243-02), has previously developed a bacteria-based delivery platform to efficiently generate and deliver short-hairpin RNAs (shRNAs) to specifically targeted tissues. This platform is not limited to shRNA delivery, and in this proposal, we will engineer it to create “SiCRISP” – a novel platform that produces and delivers all components needed for CRISPR/Cas9-directed gene editing. We will also perform proof-of-concept studies for its use in the delivery of gene editing machinery to clinically relevant tissues. By applying the key components of the SiVEC delivery platform, SiCRISP overcomes several critical shortcomings that limit the therapeutic potential of existing gene editing delivery systems: (1) it can target specific cells and tissues, and can be administered via multiple routes, e.g., intranasal, systemic, targeted injection, intraocular, intravaginal, among others; (2) it can enter cells and escape the endosome to efficiently deliver its cargo; (3) it has been experimentally confirmed as non-immunogenic, and it is not recognized by host immune cells, even after repeated delivery; (4) its mechanism does not depend on host genome integration, thus its effect is transient, reducing off-target effects, limiting toxicity, and improving safety; (5) it has no limitation on coding and delivery capacity (i.e., it simultaneously expresses and delivers all CRISPR/Cas9 components in one dose); (6) it is inexpensive to produce quickly and in large quantities; (7) as an all-in-one system, it eliminates additional manufacturing steps; (8) it can produce and deliver the components of any gene editing system (e.g., TALENs, base editors, and meganucleases) due to its coding versatility and large coding capacity. These innovative features of the SiCRISP delivery platform will help to realize the full potential of gene editing for the treatment of human disease.