A genetic engineering strategy for the improved production of the anti-helminthic

提高抗蠕虫药物生产的基因工程策略

• 批准号: 8393663
• 负责人: Jeffrey David Kittendorf
• 金额: $ 17.05万
• 依托单位: ALLUVIUM BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8393663
• 关键词: Agreement; Anabolism; Animals; Antibiotics; Antifungal Agents; Area; Biological Factors; Biotechnology; Chemicals; Clinical; DNA Sequence; Data; Development; Disease; Domestic Animals; Engineering; Evaluation; Fermentation; Fungal Chromosomes; Fungal Genome; Gene Amplification; Gene Cluster; Gene Deletion; Genes; Genetic; Genetic Engineering; Genetic Recombination; Genome; Genomics; Harvest; Health; Helminths; Human; Infection; Knock-in Mouse; Knock-out; Letters; Livestock; Malignant Neoplasms; Manufacturer Name; Marketing; Mediation; Metabolic Biotransformation; Metabolism; Methods; Michigan; Motivation; Mutagenesis; Output; Parasitic nematode; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Population; Production; Regulation; Regulator Genes; Relative (related person); Research Activity; Risk; Rural; Safety; Site; Streptomyces; Sum; System; Technology; Testing; Therapeutic; Work; analog; antimicrobial; base; chemotherapeutic agent; combat; commercial application; cost; cost effective; drug candidate; drug development; drug discovery; effective therapy; genome sequencing; improved; interest; manufacturing process; meetings; mutant; paraherquamide; paraherquamide A; phase 2 study; programs; research study; success; synthetic biology; tool; willingness

DESCRIPTION (provided by applicant): Helminth infections are responsible for severe health problems worldwide, in both human and animal populations. It is estimated that 2-3 billion people, mainly from rural and impoverished regions of the world suffer from disease due to parasitic nematodes; however, drug development and discovery efforts lack commitment to combat human infection due to the poor commercial market. In contrast, there are robust animal health discovery efforts aimed at eradicating infection in valuable domesticated livestock herds. Thus, animal health programs are often relied on to provide drug candidates for human therapeutics. Extensively tested in animals, the cost and development risk of human drugs originating from animal health are reduced. While human drug candidates still need to surpass stringent safety standards, an equal challenge is minimizing cost of goods to enable the infected population access to potent, low cost therapeutics. Accordingly, paraherquamide represents a new class of anti-helminth antibiotics that possesses broad-spectrum anti-nematodal activity and is a precursor to a recently launched animal health anti-parasitic agent 2- desoxoparaherquamide (Derquantel(tm)). Currently paraherquamide is manufactured through fermentation of the fungal strain P. simplicissimum. To maximize yields, the drug manufacturer has embarked on strain improvement programs to randomly evolve high producing mutants; however, this program has failed to achieve the desired output. Here, Alluvium proposes a modernized, genomics-driven strain improvement strategy aimed at maximizing paraherquamide production in fermentation. Specifically, Alluvium's approach is guided by the DNA sequence of the fungal genome in which key regulatory and/or biosynthetic genes responsible for paraherquamide production have been identified. Rational genetic engineering strategies, such as gene deletion or gene amplification, will be applied to key genes in efforts to rationally generate a high producing mutant phenotype. In this Phase I proposal, work is focused on rationally manipulating three genes that are predicted to regulate, both positively and negatively paraherquamide biosynthesis. In addition, Alluvium proposes to replicate the entire paraherquamide biosynthetic gene cluster within the fungal chromosome in order to boost production of anti-helminthic natural product. Following success in these initial studies, Phase II work will focus on engineering further efficiencies in paraherquamide production, including developing biocatalytic methods capable of converting paraherquamide to 2-desoxoparaherquamide as well as biotransformation tools that can be employed to generate synthetically challenging structural analogs. In sum, the strain engineering technology under development at Alluvium will result in an optimized paraherquamide manufacturing process that will lower the cost of goods required to access this commercially important anti- helminthic therapeutic, thereby enabling the potential development of a much-needed human therapeutic. PUBLIC HEALTH RELEVANCE: Helminths, especially parasitic nematodes, cause severe health problems in both humans and domesticated animals. Approximately 2-3 billion people in rural and impoverished regions are infected; however, affordable and effective treatment options are scarce. The proposed work seeks to improve upon the production of the promising anti-helminthic animal health chemotherapeutic agent, paraherquamide, so it can be generated in a more cost-effective and efficient manner, thereby stimulating further development activities for potential human treatment.

描述（由申请方提供）：蠕虫感染是全球范围内人类和动物群体严重健康问题的原因。据估计，主要来自世界农村和贫困地区的20 - 30亿人患有寄生线虫引起的疾病;然而，由于商业市场不景气，药物开发和发现工作缺乏对抗人类感染的承诺。相比之下，有强大的动物健康发现努力，旨在消除有价值的驯养牲畜群的感染。因此，经常依赖动物健康计划来为人类治疗提供候选药物。通过在动物中进行广泛的测试，降低了源自动物健康的人用药物的成本和开发风险。虽然人类候选药物仍然需要超过严格的安全标准，但同样的挑战是最大限度地降低商品成本，使感染人群能够获得有效的低成本治疗方法。因此，paraherquamide代表一类新的抗蠕虫抗生素，其具有广谱抗寄生虫活性，并且是最近推出的动物健康抗寄生虫剂2- desoxoparaherquamide（DerquantelTM）的前体。目前，通过真菌菌株P. simplicissimum的发酵来生产对海奎酰胺。为了使产量最大化，药物制造商已经开始进行菌株改良计划以随机进化高产突变体;然而，该计划未能实现期望的产量。在这里，Alluvium提出了一种现代化的、基因组学驱动的菌株改良策略，旨在最大限度地提高发酵中的对乙酰甲酰胺产量。具体而言，Alluvium的方法是由真菌基因组的DNA序列指导的，其中已经鉴定了负责paraherquamide生产的关键调控和/或生物合成基因。合理的基因工程策略，如基因缺失或基因扩增，将应用于关键基因， 合理地产生高产突变体表型。在这个第一阶段的建议，工作重点是合理地操纵三个基因，预测调节，积极和消极paraherquamide生物合成。此外，Alluvium还提出在真菌染色体内复制整个paraherquamide生物合成基因簇，以促进抗蠕虫天然产品的生产。在这些初步研究取得成功后， 工作将集中在工程上的paraherquamide生产的进一步效率，包括开发生物催化方法，能够转化paraherquamide 2-desoxoparaherquamide以及生物转化工具，可用于产生合成挑战性的结构类似物。总之，Alluvium正在开发的菌株工程技术将导致优化的对乙酰甲酰胺制造工艺，该工艺将降低获得这种商业上重要的抗蠕虫治疗剂所需的商品成本，从而能够潜在开发急需的人类治疗剂。 公共卫生相关性：蠕虫，特别是寄生线虫，对人类和家畜造成严重的健康问题。农村和贫困地区约有20亿至30亿人受到感染;然而，负担得起和有效的治疗选择很少。拟议的工作旨在改进有前途的抗蠕虫动物健康化学治疗剂paraherquamide的生产，因此可以以更具成本效益和效率的方式生产，从而刺激潜在人类治疗的进一步开发活动。