Combined RNA Production and Purification Reagents Using Liquid-Liquid Phase Separating Biopolymers

使用液-液相分离生物聚合物的组合 RNA 生产和纯化试剂

• 批准号: 10325210
• 负责人: Michael James Dzuricky
• 金额: $ 25.55万
• 依托单位: ISOLERE BIO, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2022-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325210
• 关键词: Affinity; Antibodies; Behavior; Binding; Biological; Biopolymers; Chimeric Proteins; Chromatography; Cloning; Complex; DNA; Data; Elastin; Engineering; Ensure; Enzymes; Escherichia coli; Excision; Genetic; Genetic Transcription; Goals; In Vitro; Industry Standard; Left; Length; Ligands; Liquid substance; Mainstreaming; Mass Spectrum Analysis; Measures; Messenger RNA; Methods; Methylation; Methyltransferase; Multienzyme Complexes; Optics; Pain; Patients; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plant Resins; Poly A; Poly(A) Tail; Polymerase; Polynucleotide Adenylyltransferase; Population; Price; Process; Production; Proteins; RNA; Reaction; Reagent; Recombinants; Reporter Genes; Research; Residual state; Series; Small Business Innovation Research Grant; Sodium Chloride; Speed; Tail; Technology; Temperature; Testing; Therapeutic; Transcript; Translations; Vaccination; Vaccines; Viral Vector; Vision; base; biophysical properties; cost; cost effective; cost effectiveness; design; improved; in vivo; innovation; light scattering; manufacturing process; novel; operation; polypeptide; programs; scale up; success; therapeutic development

Project Summary The goal of this Phase I SBIR project is to develop a novel purification method that is compatible with in vitro transcription (IVT)-based manufacturing of RNA for use as a therapeutic or vaccine. As RNA becomes more commonly used for pharmaceutical products, manufacturing methods must improve their scalability, speed, and cost-effectiveness. There are several pain points in RNA manufacturing. First, current affinity resins have very low capacity —only ~2mg RNA per mL— necessitating large volumes of resin at commercial scale. These resins are priced at over $25,000 per liter, rendering commercial scale production extremely costly. Second, 5’ capping of RNA – essential for in vivo function – and the subsequent removal of any residual uncapped RNA is a major bottleneck for RNA production. Isolere Bio, Inc. specializes in designing innovative purification solutions that combine a protein affinity tag that is genetically fused to an elastin-like polypeptide (ELP). This reagent is capable of temperature or salt-controlled phase separation that enables capture of a specific target regardless of its complexity or the presence of other impurities. We will develop a novel combination of biopolymers, referred to here as IVTag™, that enables simultaneous RNA sequestration into concentrated, pure liquid droplets and methylation of transcripts. Two ELP fusion proteins will be developed: one will contain an RNA-binding motif and the second an O’-methyltransferase (O’-MTase) enzyme, responsible for 5’-capping of RNA. We will then develop platform methods for simultaneous purification of RNA into droplets and high efficiency capping of the sequestered RNA. This Phase I program is designed to demonstrate compatibility of this essential IVT enzyme and complexing it with Isolere’s non-chromatographic purification methods. Key activities of the proposal include synthesizing, purifying, and testing each biopolymer fusions’ functionality and then evaluating their efficacy when applied in tandem and comparing process efficiency to the industry standard method. If successful, additional functionality of the RNA micro-crucible will be developed in Phase II in the form of a T7 polymerase-ELP fusion (for initial transcription of a DNA template) and a Poly-A polymerase-ELP fusion (for adding a 3’ poly-A tail to transcripts). Our vision is to ultimately create a fully continuous RNA manufacturing process in smart droplets. These smart droplets are expected to support a straightforward, scalable, and cost-effective manufacturing workflow with micro-crucibles capable of carrying out a complex series of processing steps. Because the micro- crucible is volume independent, the scale-up difficulties associated with affinity resins are eliminated. In addition, with other applications of Isolere’s technology, including to antibodies and viral vectors, the ELP fusion reagents have proven to be easily manufacturable and re-usable for multiple purification cycles, reducing costs of goods significantly compared to affinity resins. The scalability, robust performance, and cost-effectiveness of the proposed product is an excellent fit for accelerating the development of therapeutic RNA candidates from bench scale discovery through large-scale manufacturing of commercial products.

项目摘要 该阶段I SBIR项目的目标是开发一种与体外兼容的新型纯化方法 基于转录（IVT）的RNA制造作为治疗或疫苗。随着RNA变得更多 制造方法通常用于药品，必须提高其可扩展性，速度和 成本效益。 RNA制造中有几个疼痛点。首先，当前的亲和力树脂非常 低容量 - 每毫升的〜2mg RNA - 在商业尺度上覆盖大量树脂。这些树脂 价格的价格超过25,000美元，使商业规模的生产非常昂贵。第二，5'上限 RNA的 - 体内功能必不可少的 - 随后去除任何残留的无封闭RNA是主要的 用于RNA生产的瓶颈。 Inlyere Bio，Inc。专门设计创新的纯化解决方案 结合蛋白亲和力标签，该蛋白亲和力标签与弹性蛋白样的多肽（ELP）结合在一起。该试剂有能力 温度或盐控制相的分离，可以捕获特定靶标的 复杂性或其他杂质的存在。我们将开发出一种新颖的生物聚合物组合，称为 在这里，作为IVTAG™，可以同时将RNA隔离为浓缩物，纯液滴和 转录本的甲基化。将开发两个ELP融合蛋白：一个将包含一个RNA结合基序 第二种O'-甲基转移酶（O'-MTase）酶，负责R​​NA的5'限制。然后我们会 开发平台方法，将RNA简单纯化为液滴和高效上限 隔离的RNA。该阶段I计划旨在证明这种基本IVT酶的兼容性 并将其与Iselere的非色谱纯化方法进行复合。该提案的关键活动包括 合成，净化和测试每个生物聚合物融合的功能，然后在评估其有效性时 在同时使用并将过程效率与行业标准方法进行比较。如果成功，请额外 RNA Micro-Crabucrable的功能将以T7聚合酶-ELP融合的形式在II期中开发 （用于DNA模板的初始转录）和Poly-A聚合酶-ELP融合（用于将3'poly-A尾巴添加到 成绩单）。我们的愿景是最终在智能液滴中创建完全连续的RNA制造过程。 这些智能液滴有望支持直接，可扩展和具有成本效益的制造业 具有小型疑惑的工作流，能够执行一系列复杂的处理步骤。因为微型 坩埚是独立的，与亲和树脂相关的规模难度被消除。此外， 借助Iselere技术的其他应用，包括抗体和病毒向量，ELP Fusion试剂 事实证明，对于多个净化周期，很容易制造和重新使用，降低了商品的成本 与亲和力树脂相比显着。可扩展性，稳健性能和成本效益 拟议的产品非常适合加速替补席的治疗性RNA候选物 通过大规模制造商业产品进行扩展发现。