ISSLS prize in basic science 2021: a novel inducible system to regulate transgene expression of TIMP1.
ISSLS prize in basic science 2021: a novel inducible system to regulate transgene expression of TIMP1.
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2021年ISSLS基础科学奖：一种调节TIMP1转基因表达的新型诱导系统
DOI：
10.1007/s00586-021-06728-0
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发表时间：
2021-05
期刊：
影响因子：
--
通讯作者：
Vo NN
中科院分区：
文献类型：
--
作者：
Han Y;Ouyang Z;Wawrose RA;Chen SR;Hallbaum M;Dong Q;Dando E;Tang Y;Wang B;Lee JY;Shaw JD;Kang JD;Sowa GA;Vo NN
目的炎症和氧化应激上调基质金属蛋白酶（MMP）活性，导致椎间盘退变（IDD）。利用人组织金属蛋白酶1抑制剂（hTIMP1）进行基因治疗可以有效治疗动物模型中的IDD。然而，持续不受管制的转基因表达可能有负面的副作用。我们构建了一种重组腺相关病毒（AAV）基因载体AAV- nfκ b -hTIMP1，该载体在逆境条件下仅表达hTIMP1基因。方法用AAV-CMV-hTIMP1或AAV-NFκB-hTIMP1转染或转导兔椎间盘细胞。用IL-1β选择性处理椎间盘细胞。核易位证实NFκB活化。RT-PCR和ELISA分别检测hTIMP1 mRNA和蛋白的表达。MMP的活性是通过裂解荧光底物来测定的。结果IL-1β刺激可激活NFκB，表明IL-1β是炎症应激的替代物。与未刺激的细胞相比，用IL-1β刺激AAV-NFκB-hTIMP1细胞增加了hTIMP1的表达。无论IL-1β是否刺激，AAV-CMV-hTIMP1细胞均表现出高水平的hTIMP1表达。在IL-1β刺激的AAV-NFκB-hTIMP1细胞和AAV-CMV-hTIMP1细胞中，hTIMP1的表达具有可比性。与基线水平或暴露于IL-1β的细胞相比，AAV-NFκB-hTIMP1细胞的MMP活性降低。结论aav - nfκ b - htimp1是一种新型的诱导型转基因传递系统。NFκB调控元件确保hTIMP1仅在炎症中表达，这是IDD发展的核心。与之前的诱导系统不同，AAV-NFκB-hTIMP1构建依赖于内源性因子，从而最大限度地减少了组成型转基因过表达引起的潜在副作用。它还可以防止在不需要治疗的细胞中产生不必要的转基因产物。
PurposeInflammatory and oxidative stress upregulates matrix metalloproteinase (MMP) activity, leading to intervertebral disc degeneration (IDD). Gene therapy using human tissue inhibitor of metalloproteinase 1 (hTIMP1) has effectively treated IDD in animal models. However, persistent unregulated transgene expression may have negative side effects. We developed a recombinant adeno-associated viral (AAV) gene vector, AAV-NFκB-hTIMP1, that only expresses the hTIMP1 transgene under conditions of stress.MethodsRabbit disc cells were transfected or transduced with AAV-CMV-hTIMP1, which constitutively expresses hTIMP1, or AAV-NFκB-hTIMP1. Disc cells were selectively treated with IL-1β. NFκB activation was verified by nuclear translocation. hTIMP1 mRNA and protein expression were measured by RT-PCR and ELISA, respectively. MMP activity was measured by following cleavage of a fluorogenic substrate.ResultsIL-1β stimulation activated NFκB demonstrating that IL-1β was a surrogate for inflammatory stress. Stimulating AAV-NFκB-hTIMP1 cells with IL-1β increased hTIMP1 expression compared to unstimulated cells. AAV-CMV-hTIMP1 cells demonstrated high levels of hTIMP1 expression regardless of IL-1β stimulation. hTIMP1 expression was comparable between IL-1β stimulated AAV-NFκB-hTIMP1 cells and AAV-CMV-hTIMP1 cells. MMP activity was decreased in AAV-NFκB-hTIMP1 cells compared to baseline levels or cells exposed to IL-1β.ConclusionAAV-NFκB-hTIMP1 is a novel inducible transgene delivery system. NFκB regulatory elements ensure that hTIMP1 expression occurs only with inflammation, which is central to IDD development. Unlike previous inducible systems, the AAV-NFκB-hTIMP1 construct is dependent on endogenous factors, which minimizes potential side effects caused by constitutive transgene overexpression. It also prevents the unnecessary production of transgene products in cells that do not require therapy.
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影响因子：
4.9
作者：
Le Maitre, Christine L;Hoyland, Judith A;Freemont, Anthony J
通讯作者：
Freemont, Anthony J
单细胞 NF-kappaB 动力学揭示了数字激活和模拟信息处理。
影响因子：
64.8
作者：
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影响因子：
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作者：
Bartfeld S;Hess S;Bauer B;Machuy N;Ogilvie LA;Schuchhardt J;Meyer TF
通讯作者：
Meyer TF
影响因子：
3
作者：
DEYO, RA;TSUIWU, YJ
通讯作者：
TSUIWU, YJ
影响因子：
3.1
作者：
DePalma, Michael J.;Ketchum, Jessica M.;Saullo, Thomas
通讯作者：
Saullo, Thomas