Human genetic supplementation without donor DNA or a DNA break

摘要 人类基因组工程作为一种医疗保健战略已被广泛预期，但目前的技术 不太可能提供安全、有效和广泛有用的转基因导入所必需的实施方案。 来完成基因治疗的下一个飞跃基于CRISPR的转基因整合方法 有主要的障碍，包括需要供体DNA的交付，DNA的倾向， 非特异性整合，和低效率的修复同源重组相对草率 断裂DNA末端的重新连接。同样严重限制的事实是，缓慢增殖的细胞很少在一个细胞中生长。 细胞周期阶段有利于同源重组，并且仅仅存在DNA断裂可能是有毒的。 腺相关病毒引入转基因的替代方法也具有局限性，其中 其他包括病毒衣壳允许的小转基因大小和工程病毒的挑战 进入不同的细胞类型。仍然需要一种无诱变性、无毒的方法， 将基因导入人类基因组。许多功能丧失性疾病的治疗取决于这种缺失 技术.而且，只有转基因导入提供了蛋白质表达的非天然控制的机会， 异构体选择性和无数其他临床有用的结果。 目前开发转基因导入技术的努力中完全缺少的是一种利用 基因插入策略在真核生物中广泛存在：cDNA合成。祖先， 进化持续型真核细胞LINE/non-LTR逆转录元件通过缺口引物反向整合 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特异性 以逆转录元件3' UTR为模板的RNA转录物。目标所需的生化活性 位点选择、精确定位切口的引入和cDNA合成通过单一蛋白质进行。 侧接逆转录元件基因组的5'和3'区的任何RNA序列应该与有利的互补序列组装。 修饰的逆转录元件蛋白，然后该RNP将寻找其天然插入位点。因为几 LINE/非LTR逆转录元件家族靶向高度保守、重复序列，在整个序列中保持不变 对于多细胞真核生物，不需要重新设计这些逆转录因子蛋白的DNA位点特异性， 虽然这可能会成为兴趣承担。非LTR回溯元素的简单架构 用于开发一种用治疗基因补充人类基因组的方法， 冲击这种方法的新奇要求不断创新，并承担着很高的失败风险 目的是将能够转基因导入人细胞的工程化RNP递送。成功的这项 该战略将为功能丧失疾病的治疗带来一种新的模式。

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.