Directed evolution of a sequence-specific targeting technology for therapeutic gene delivery to the human genome.

用于将治疗基因递送至人类基因组的序列特异性靶向技术的定向进化。

• 批准号: 10400161
• 负责人: Jesse B Owens
• 金额: $ 58.63万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10400161
• 关键词: Address; Bacteria; Bacteriophages; Biological Assay; Cell division; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; DNA; DNA Binding; DNA Integration; Data; Development; Directed Molecular Evolution; Disease; Drops; Enzymes; Escherichia coli; Evolution; Factor IX; Gene Delivery; Gene Targeting; Genes; Genetic Diseases; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Hemophilia B; Hepatocyte; Human; Human Cell Line; Human Genome; Immune response; Individual; Insertional Mutagenesis; Integrase; Integration Host Factors; Interphase Cell; Intervention; Laboratories; Lead; Liver; Location; Malignant Neoplasms; Microbubbles; Modeling; Mus; Mutate; Mutation; Pathway interactions; Patients; Proteins; Publishing; Reporter; Research; Research Personnel; Risk; Serine; Site; Specificity; System; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Transgenes; Variant; Virus; base; detection platform; experimental study; flexibility; gene correction; gene replacement; gene therapy; genome editing; genome-wide analysis; improved; innovation; mouse model; new technology; novel; nuclease; plasmid DNA; pre-clinical research; repaired; therapeutic gene; therapeutic transgene; therapy outcome; tool; transgene expression; ultrasound; vector

PROJECT SUMMARY Unnecessary risk by random insertional mutagenesis and uncontrolled expression of inserted transgenes represent critical issues with current non-targeted gene therapy approaches. Homology directed repair (HDR) can target inserts but is inefficient, especially in non-dividing cells or if the donor DNA is large. Currently, a system for efficiently inserting a therapeutic gene to a known sequence is critically needed. To address these challenges, the applicants have developed a novel system for DNA integration by evolving integrase enzymes to insert DNA of flexible size at a desired sequence in the human genome. The long-term goal is to develop clinical therapies that use insertional vectors to treat genetic disease. The central hypothesis is that directed evolution will produce an integrase capable of targeting a single attP site in the genome without off-target insertion. To demonstrate translational applicability, the evolved integrase will be assayed for delivery of the therapeutic Factor IX gene to the liver of hemophilic mice. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratories clearly demonstrating that their directed evolution approach successfully improves integrase specificity. The rationale is that, development of this new tool will allow researchers and clinicians to deliver therapeutic transgenes to a single, known sequence. This would overcome risks of insertional mutagenesis and facilitate predictable transgene expression. In Aim 1, directed evolution will be used to repeatedly evolve and select for variants with improved targeting specificity in order to generate integrases active on a single sequence in the genome. In Aim 2, evolved integrases will be screened for activity in human cell lines and a genome-wide analysis of possible off-targets will be performed. In Aim 3, the site- specific integrase will be delivered to the liver of a mouse model of Hemophilia B. The therapeutic potential of treating Hemophilia B with this technology will be assessed. The project is highly innovative because it uses an advanced continuous directed evolution system that is orders of magnitude faster than traditional approaches and has never been applied to improving integrase vectors. These sequence targeting vectors will be combined with a non-invasive, tissue-specific delivery approach for the first time. The proposed research is significant because it develops a tool capable of safely and efficiently directing therapeutic genes to a desired sequence in the genome without negative off-target consequences. Patients suffering from genetic diseases frequently have a variety of distinct mutations. This technology could be used to insert a corrected gene copy to treat disease, irrespective of an individual's mutation. Complex disorders could be treated by delivering multiple genes or whole biosynthetic pathways. In order to demonstrate a therapeutic application, Factor IX will be delivered to mice to model a treatment for Hemophilia B. Significantly, because the size and sequence of the inserted DNA is flexible, this platform technology is adaptable to preclinical research applications as well as potential treatments of any disease requiring gene replacement.

项目摘要 随机插入诱变和插入转基因的不受控制表达造成的不必要风险 代表了当前非靶向基因治疗方法的关键问题。同源定向修复 可以靶向插入物，但效率低下，特别是在非分裂细胞中或如果供体DNA很大。现时 迫切需要将治疗基因有效地插入已知序列的系统。解决这些 为了克服这些挑战，申请人开发了一种通过进化整合酶来进行DNA整合的新系统 在人类基因组中的所需序列处插入灵活大小的DNA。长期目标是发展 使用插入载体治疗遗传疾病的临床疗法。核心假设是， 进化将产生能够靶向基因组中单个attP位点而不脱靶的整合酶 插入。为了证明翻译适用性，将测定进化的整合酶的递送。 治疗因子IX基因的血友病小鼠的肝脏。这一假设是根据以下事实提出的： 申请人实验室产生的初步数据清楚地表明， 该方法成功地提高了整合酶的特异性。理由是，开发这一新工具将允许 研究人员和临床医生将治疗性转基因递送到单个已知序列。这将克服 插入诱变的风险，并促进可预测的转基因表达。在目标1中，定向进化 用于重复进化和选择具有改进的靶向特异性的变体， 在基因组中的单个序列上有活性的整合酶。在目标2中，将筛选进化的整合酶的活性 并将对可能的脱靶进行全基因组分析。在目标3中，网站- 将特异性整合酶递送至血友病B小鼠模型的肝脏。的治疗潜力 将评估使用该技术治疗血友病B的效果。该项目具有很高的创新性，因为它使用了 先进的连续定向进化系统，比传统方法快几个数量级 并且从未应用于改进整合酶载体。这些序列靶向载体将被组合 第一次采用非侵入性的组织特异性输送方法。所提出的研究是有意义的 因为它开发了一种能够安全有效地将治疗基因导向所需序列的工具， 基因组没有负面脱靶后果。患有遗传性疾病的患者经常有 各种不同的突变这项技术可以用来插入一个正确的基因拷贝来治疗疾病， 不管个体的突变如何。复杂的疾病可以通过递送多个基因或整个 生物合成途径为了证明治疗应用，将因子IX递送至小鼠， 血友病B的治疗模式。值得注意的是，由于插入DNA的大小和序列是灵活的， 该平台技术适用于临床前研究应用以及任何疾病的潜在治疗。 需要基因替换的疾病