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Promoterless gene targeting without nucleases ameliorates haemophilia B in mice

机译：无核酸酶的无启动子基因靶向改善小鼠血友病B

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摘要

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform re-combinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor Ⅸ (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9vector into neonatal and adult mice and achieved on-target integration into ～0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.
机译：位点特异性基因添加可以使基因治疗中稳定的转基因表达。在可能的情况下，这比使用混杂整合的载体更为可取，后者有时与克隆扩增和肿瘤发生有关。可以诱导靶向基因组编辑的高速率的位点特异性核酸内切酶在生物发现和基因治疗中的应用越来越广泛。但是，仍然存在两个安全隐患：核酸内切酶相关的不良反应，无论是靶上还是靶外；和启动子整合引起的癌基因激活，即使没有核酸酶也是如此。在这里，我们进行重组重组腺相关病毒（rAAV）介导的无核酸酶的无启动子基因靶向，并证明了血友病B小鼠出血素质的改善。特别地，我们将无启动子的人凝血因子Ⅸ（F9）基因靶向肝表达的小鼠白蛋白（Alb）基因座。 F9与前面的2A肽编码序列一起被靶向整合到Alb终止密码子的上游。当F9在DNA和RNA水平上与Alb融合时，通过核糖体跳跃合成了两种不同的蛋白质。因此，F9表达与健壮的肝白蛋白表达相关，而不会破坏它。我们向新生小鼠和成年小鼠注射了AAV8-F9载体，并实现了约0.5％肝细胞白蛋白等位基因的靶标整合。我们确定F9仅由目标整合产生，核糖体跳跃非常高效。获得的F9血浆水平稳定在正常水平的7-20％，并且治疗的F9缺陷型小鼠的凝血时间正常。总之，将转基因整合为2A融合至高度表达的内源基因可以消除对核酸酶和/或载体携带的启动子的需求。该方法可通过大大减少脱靶作用，同时仍提供整合治疗水平的表达，从而在婴儿和成人中实现安全有效的基因靶向。

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  • 来源
    《Nature》 |2015年第7534期|360-364|共5页
  • 作者

    A. Barzel; N. K. Paulk; Y. Shi; Y. Huang; K. Chu; F. Zhang; P. N. Valdmanis; L. P. Spector; M. H. Porteus; K. M. Gaensler; M. A. Kay;

  • 作者单位

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Department of Medicine, Box 1270, UCSF, San Francisco, California 94143-1270, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

    Department of Pediatrics, 269 Campus Drive, Lorry Lokey Stem Cell Research Building, Room G3045, Stanford, California 94305-5164, USA;

    Department of Medicine, Box 1270, UCSF, San Francisco, California 94143-1270, USA;

    Departments of Pediatrics and Genetics, 269 Campus Drive, CCSR Building, Room 2105, Stanford, California 94305-5164, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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