Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.
Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.
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DOI：
10.1038/nature13864
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发表时间：
2015-01-15
期刊：
影响因子：
64.8
通讯作者：
Kay, M. A.
中科院分区：
文献类型：
--
作者：
Barzel, A.;Paulk, N. K.;Shi, Y.;Huang, Y.;Chu, K.;Zhang, F.;Valdmanis, P. N.;Spector, L. P.;Porteus, M. H.;Gaensler, K. M.;Kay, M. A.
特定于特定的基因可以在可能的情况下进行稳定的转化表达，这比有时的使用载体相比，这是有时与克隆膨胀和肿瘤发生有关的载体。编辑发现在生物发现和基因治疗中的应用增加。不良影响，无论是启动子积分而引起的，即使没有核能，我们都会进行重组腺相关病毒（RAAV）介导的启动子基因，并证明了出血性核心的降低特别是，我们将无启动子凝血因子IX（HF9）基因靶向肝脏表达的白蛋白（ALB）基因座。 2A肽编码序列，仅在ALB停止密码子上游，而HF9在DNA和RNA水平上融合在一起，两个单独的蛋白质通过核糖体跳过。白蛋白的表达而无需破坏它。肝细胞。我们确定HF9仅是通过靶标的整合而产生的，核糖体跳动效率很高。转基因整合作为2A融合到高度表达的内源基因可能会消除对核和/或载体启动子的需求。在婴儿和成年人中的有效基因靶向，通过大大减少脱靶效应，同时仍然可以从整合中提供治疗水平的表达水平。
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: (1) endonuclease-associated adverse effects, both on and off-target; and (2) oncogene activation caused by promoter integration, even without nucleases. Here, we perform recombinant 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 (hF9) gene to the liver-expressed albumin (Alb) locus. hF9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While hF9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, hF9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-hF9 vector into neonatal and adult mice and achieved on-target integration into ~0.5% of the albumin alleles in hepatocytes. We established that hF9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable hF9 plasma levels at 7–20% of normal were obtained, and treated factor IX 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.
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影响因子：
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作者：
Grimm, Dirk;Streetz, Konrad L.;Kay, Mark A.
通讯作者：
Kay, Mark A.