Human genetic supplementation without donor DNA or a DNA break

无需供体 DNA 或 DNA 断裂的人类基因补充

• 批准号: 10012227
• 负责人: Kathleen Collins
• 金额: $ 111.48万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10012227
• 关键词: 3' Untranslated Regions; 5' Flanking Region; Architecture; Biochemical; Capsid; Cell Cycle; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; DNA; DNA biosynthesis; DNA delivery; Dependovirus; Development; Disease; Engineering; Eukaryota; Evolution; Failure; Family; Genes; Genome; Genome engineering; Goals; Healthcare; Human; Human Genetics; Human Genome; Modality; Outcome; Pathology; Phase; Positioning Attribute; Proliferating; Protein Isoforms; Proteins; RNA; RNA Sequences; RNA chemical synthesis; Repetitive Sequence; Retroelements; Reverse Transcription; Site; Specificity; Supplementation; Techniques; Technology; Therapeutic; Transcript; Transgenes; Virus; base; cell type; gene therapy; high risk; homologous recombination; innovation; interest; loss of function; non-Native; protein expression; repaired; success; uptake

ABSTRACT Human genome engineering has widely anticipated promise as a healthcare strategy, but current technologies are unlikely to provide the safe, efficient, and broadly useful implementation of transgene introduction essential to complete the next big leap forward for gene therapy. CRISPR-based approaches for transgene integration have major impediments, including the need for donor DNA delivery, the propensity of that DNA to undergo non-specific integration, and the low efficiency of repair by homologous recombination relative to sloppy rejoining of the broken DNA ends. Also severely limiting is the fact that slowly proliferating cells are rarely in a cell cycle phase favorable for homologous recombination, and just the presence of a DNA break can be toxic. The alternative approach of adeno-associated virus introduction of a transgene also has limitations, among others including the small transgene size permitted by the virus capsid and the challenges of engineering virus uptake into different cell types. It remains an unmet need to have a non-mutagenic, non-toxic approach for gene introduction to the human genome. Therapy for many loss-of-function pathologies hinges on this missing technology. Also, only transgene introduction offers the opportunity for non-native control of protein expression, isoform selectivity, and myriad other clinically useful outcomes. Starkly missing from current efforts to develop transgene introduction techniques is an approach exploiting the gene insertion strategy widespread endogenously across eukaryotes: cDNA synthesis. The ancestral, evolutionarily persistent type of eukaryotic LINE/non-LTR retroelement integrates by nick-primed reverse transcription that is rigorous both it its sequence specificity of target site selection and in its specificity for use of an RNA transcript with the retroelement 3’ UTR as template. The biochemical activities required for target site selection, introduction of precisely positioned nick, and cDNA synthesis are carried out by a single protein. Any RNA sequence flanked by 5’ and 3’ regions of the retroelement genome should assemble with a favorably modified retroelement protein, and this RNP would then seek its native insertion site. Because several LINE/non-LTR retroelement families target highly conserved, repetitive sequences invariant across multicellular eukaryotes, there is no need to re-engineer DNA site-specificity of these retroelement proteins, although that may become of interest to undertake. The simple architecture of the non-LTR retroelements begs to be exploited for developing an approach to human genome supplementation with genes of therapeutic impact. The novelty of this approach demands continuous innovation and obliges high risk of failure to reach the goal of delivering an engineered RNP capable of transgene introduction into human cells. Success of this strategy would usher in a new modality of therapeutic treatment for loss-of-function diseases.

抽象的 人类基因组工程作为一种医疗保健策略被广泛期待，但目前的技术 不太可能提供安全、高效和广泛有用的转基因导入实施方案 完成基因治疗的下一个大飞跃。基于 CRISPR 的转基因整合方法 存在重大障碍，包括需要供体 DNA 递送、该 DNA 的倾向 非特异性整合，同源重组修复效率相对马虎而言较低 断裂的 DNA 末端重新连接。同样严重的限制是，缓慢增殖的细胞很少处于同一状态。 有利于同源重组的细胞周期阶段，仅 DNA 断裂的存在就可能具有毒性。 腺相关病毒引入转基因的替代方法也有局限性，其中 其他包括病毒衣壳允许的小转基因尺寸以及工程病毒的挑战 摄取到不同的细胞类型中。对于非诱变、无毒的方法，仍然是一个未满足的需求 基因引入人类基因组。许多功能丧失性疾病的治疗取决于这种缺失 技术。此外，只有转基因引入才能提供非天然控制蛋白质表达的机会， 同种型选择性，以及无数其他临床有用的结果。 当前开发转基因导入技术的努力中明显缺少的是一种利用方法 真核生物内源性广泛存在的基因插入策略：cDNA 合成。祖传的， 真核细胞 LINE/非 LTR 逆转录元件的进化持久型通过切口引物反向整合 转录是严格的，无论是其靶位点选择的序列特异性还是其使用的特异性 以逆转录元件 3' UTR 作为模板的 RNA 转录本。目标所需的生化活性 位点选择、精确定位切口的引入以及 cDNA 合成均由单一蛋白质完成。 任何侧翼为逆转录元件基因组 5' 和 3' 区域的 RNA 序列都应以有利的方式组装 修饰逆转录元件蛋白，然后该 RNP 将寻找其天然插入位点。因为几个 LINE/非 LTR 逆转录元件家族靶向高度保守、跨区域不变的重复序列 多细胞真核生物，无需重新设计这些逆转录元件蛋白的 DNA 位点特异性， 尽管这可能会引起人们的兴趣。非 LTR 逆向元件的简单架构需要 用于开发一种用治疗基因补充人类基因组的方法 影响。这种方法的新颖性需要持续创新，并且存在很高的失败风险 目标是提供能够将转基因引入人类细胞的工程化 RNP。此次成功 该策略将为功能丧失性疾病带来一种新的治疗方式。