Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice.
Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice.
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DOI：
10.1186/s13024-016-0128-x
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发表时间：
2016-09-06
影响因子：
15.1
通讯作者：
Isalan M
中科院分区：
文献类型：
--
作者：
Agustín-Pavón C;Mielcarek M;Garriga-Canut M;Isalan M
合成锌指(ZF)蛋白可以针对所需的DNA序列，是基因治疗的有用工具。我们最近开发了一种ZF转录抑制因子(ZF-KOX1)，能够与亨廷顿病(HD)基因中扩展的DNA CAG重复序列结合。该ZF在HD小鼠模型中强烈抑制突变HTT的表达，并延迟神经症状(扣带)长达3周。在目前的工作中，我们试图开发一种长期的脑内单次注射基因治疗方法。由于非自身蛋白可以引发免疫和炎症反应，我们设计了一种宿主匹配的ZF-KOX1类似物(称为mZF-KRAB)，结合rAAV载体传递，可以更安全地治疗小鼠。我们还测试了神经元特异性烯醇酶启动子(PNSE)，据报道它能够实现长期转基因表达，以确定在脑内注射AAV后是否可以观察到HTT抑制长达6个月。在rAAV载体传递后，我们发现非自身蛋白在大脑中诱导了显著的炎症反应，这与以前的研究一致。具体地说，在非宿主匹配的ZF-KOX1或GFP治疗后4周和6周，小胶质细胞分别被激活，并伴有中度神经元丢失。相反，宿主匹配的mZF-KRAb没有引起这些影响。然而，我们发现使用pCAG启动子(CMV早期增强子元件和鸡β-肌动蛋白启动子)在注射后6周导致ZF表达显著减少。因此，我们测试了一种新的非病毒启动子，看看宿主适应的ZF表达是否可以持续更长时间。在双侧脑室注射1010病毒粒子3周后，用神经元特异性烯醇化酶启动子增强子(Eno2，大鼠)向mZF-KRAb表达mZF-KRAb后，突变型HTT在全脑中的抑制率高达77%。重要的是，在12周和24周后，仍分别检测到48%和23%的表达，这表明更长期的影响是可能的。宿主适应的ZF-AAV载体显示出较低的毒性，非病毒型pNSE启动子改善了ZF蛋白的长期表达和靶基因的抑制。本文提出的优化结构具有治疗HD的潜力。本文的在线版本(doi：10.1186/s130240160128-x)包含补充材料，授权用户可以使用。
Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington’s disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain. Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain. After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010 virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible. Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD. The online version of this article (doi:10.1186/s13024-016-0128-x) contains supplementary material, which is available to authorized users.
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影响因子：
16.2
作者：
Kordasiewicz HB;Stanek LM;Wancewicz EV;Mazur C;McAlonis MM;Pytel KA;Artates JW;Weiss A;Cheng SH;Shihabuddin LS;Hung G;Bennett CF;Cleveland DW
通讯作者：
Cleveland DW
影响因子：
46.9
作者：
Isalan, M;Klug, A;Choo, Y
通讯作者：
Choo, Y
影响因子：
4
作者：
Agustin-Pavon, Carmen;Isalan, Mark
通讯作者：
Isalan, Mark
DOI：
10.1073/pnas.98.4.1437
发表时间：
2001-02-13
影响因子：
11.1
作者：
Moore, M;Klug, A;Choo, Y
通讯作者：
Choo, Y
DOI：
10.1073/pnas.041616998
发表时间：
2001-02-13
影响因子：
11.1
作者：
Abrink, M;Ortiz, JA;Losson, R
通讯作者：
Losson, R