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: 10687195
• 负责人: Kathleen Collins
• 金额: $ 112.35万
• 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/10687195
• 关键词: 3' Untranslated Regions; 5' Flanking Region; Architecture; Biochemical; CRISPR/Cas technology; Capsid; Cell Cycle; Cell Proliferation; Cells; Clinical; 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; Protein Isoforms; Proteins; RNA; RNA Sequences; RNA chemical synthesis; Repetitive Sequence; Retroelements; Reverse Transcription; Site; Specificity; Supplementation; Techniques; Technology; Therapeutic; Transcript; Transgenes; Virus; 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 人类基因组工程已广泛期待成为医疗策略的希望，但是当前的技术 不太可能提供安全，高效且广泛有用的转换简介实施 完成基因治疗的下一个大型飞跃。基于CRISPR的转基因整合方法 有重大障碍，包括供体DNA的需求，该DNA的承诺会经历 非特异性整合，以及相对于草率的同源重组的修复效率低 破裂的DNA末端的重新加入。严重限制的事实是，缓慢增殖的细胞很少在 有利于同源重组的细胞循环阶段，仅存在DNA断裂可能是有毒的。 腺相关病毒引入转化的另一种方法也有局限性 其他包括病毒capsid允许的小变化尺寸和工程病毒的挑战 吸收不同的细胞类型。它仍然需要采用非毒素，无毒的方法 人类基因组的基因介绍。许多功能丧失病理的疗法取决于这种缺失 技术。同样，只有转化引入为非母体控制蛋白质表达提供了机会， 同工型选择性以及其他临床上有用的结果。 目前为开发转型介绍技术而缺少的方法是一种利用的方法 基因插入策略跨真核生物内源性宽度：cDNA合成。祖先， 在进化上持续的真核线/非LTR retroment，由nick-trimed反向集成 转录是其目标位点选择的序列特异性以及其特异性的严格性 带有retroement 3'utr作为模板的RNA转录本的。目标所需的生化活动 位点选择，精确定位的尼克的引入和cDNA合成是由单个蛋白质进行的。 重新元基因组的5'和3'区域两侧的任何RNA序列都应与有利的 经过修改的恢复蛋白质，然后该RNP将寻求其天然插入位点。因为几个 线/非LTR返回元素的目标是高度保守的重复序列不变 多细胞真核生物，无需重新设计这些恢复元素的DNA位点特异性， 尽管这可能会引起人们的关注。非LTR重新元素的简单结构乞讨 通过使用治疗基因来开发补充人类基因组的方法来利用 影响。这种方法的新颖性需要持续的创新，并义务不可能达到的高风险 提供能够转化为人类细胞的工程RNP的目的。成功的成功 策略将引入新的治疗方法，以解决功能丧失疾病。