Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis
Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis
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Crispr/Cas9 改造小鼠 Calr 基因中的 61bp 缺失导致血小板增多症
DOI：
10.1182/blood.v128.22.4274.4274
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发表时间：
2016
期刊：
影响因子：
20.3
通讯作者：
S. Constantinescu
中科院分区：
文献类型：
--
作者：
Thomas Balligand;Y. Achouri;I. Chachoua;C. Pecquet;J. Defour;S. Constantinescu
在一组患有骨髓增生性肿瘤(MPN)的患者中，钙网蛋白(CALR)外显子9移码突变被认为是导致原发性血小板增多症(ET)或原发性骨髓纤维化(PMF)的原因(1，2)。最常见的突变是52个碱基的缺失(del52，类型1突变)和5个碱基的TTGTC插入(ins5，类型2突变)。在这些患者中，突变状态几乎总是杂合子。我们的团队和合作者最近发现，致病突变的CALR蛋白需要与血小板生成素受体(TpoR)相互作用并激活，以激活JAK-STAT途径(3，4)。到目前为止，还没有发表过这些疾病的敲击小鼠模型。在这个摘要中，我们展示了我们如何成功地创建了这样一个模型。我们已经证明，小鼠CALR突变蛋白的行为与它们的人类对应蛋白一样(5)。具体地说，del52、ins5和del61(61bp缺失，1型)CALR突变能够转化表达血小板生成素受体(TpoR)的BA/F3细胞(小鼠前B淋巴细胞通常依赖IL-3生长)，并使它们不依赖细胞因子。重要的是，我们还使用广泛采用的CRISPR/Cas9系统突变了BA/F3基因组，以创建CALR外显子9的61个碱基的缺失。这也成功地转化了BA/F3细胞，表明突变的CALR蛋白的内源性表达水平足以在体外诱导表型。现在，我们使用相同的方法，将相同的CRISPR/Cas9构建体注射到C57BL/6J小鼠受精卵中，以在小鼠CALR基因中产生相同的61个碱基的缺失。在这一过程中出生的46只幼崽中，有一只雄性幼崽是61个碱基对缺失的杂合子。通过体外受精，我们获得了杂合子CALRdel61/WT仔猪。经过杂交后，我们分析了12只CALRdel61/WT雄性和12只CALRWT/WT雄性(产仔)在三个不同的时间点(15、18和22周龄)的血液，发现CALRdel61/WT小鼠的循环血小板水平显著高于CALRdel61/WT小鼠。相反，在所有时间点，两组之间的红细胞和白细胞数量都是相同的。我们进一步证明，突变的CALR蛋白在杂合子状态下的表达足以诱导小鼠巨核细胞的异常增殖并在体内形成ET表型。通过对表型、骨髓和脾病理的动态观察(骨髓增殖和纤维化检查)，可以与CALR突变MPNS的逆转录病毒小鼠模型进行比较，并与人类疾病的已知特征进行比较。我们模型的唯一局限性是CALRdel61突变是父母获得的，并且在整个生物体中广泛存在。在这个新的模型中，我们的目标是测试各种药物预防或治疗MPN表型的效率，例如Ruxolitinib，一种JAK2-1抑制剂，已经在临床上用于CALR突变的MPN患者。我们现在也有了一种方法来产生大量的CALRdel61/WT骨髓细胞，以广泛地研究CALRdel61/WT突变在不同造血阶段的致癌特性。如果在体内产生CALRdel61的纯合子突变状态，研究它也将是非常有意义的。因此，我们的系统将阐明带负电的CALR尾巴的重要性，以及新的带正电的尾巴对骨髓增殖的影响。参考文献1.Klampfl T，Gisslinger H，Harutyunyan AS，Nivarthi H，Rumi E，Milosevic JD等。N Engl J Med.2013年12月10日；369(25)：2379-90。2.Nangalia J，Massie CE，Baxter EJ，Nice FL，Gundem G，Wedge DC等。N Engl J Med.2013年12月10日；369(25)：2391-405。3.Chachoua I，Pecket C，El-Khoury M，Nivarthi H，Albu RI，Marty C等人。血。2015年12月14日；10.1182/血液-2015年-11-681932。4.Marty C、Pecquet C、Nivarthi H、Elkhoury M、Chachoua I、Tulliez M等人。血。2015年11月25日；10.1182/血液-2015年11月-679571。5.Balligand T，Achouri Y，Pecket C，Chachoua I，Nivarthi H，Marty C等人。白血病。2016年2月29；10.1038/leu 2016.47.披露条件：TEVA：董事会或咨询委员会成员资格，发言人局；诺华公司：董事会或咨询委员会成员资格，发言人局；夏尔：董事会或咨询委员会成员资格，发言人局；个人遗传：董事会或咨询委员会成员资格，发言人局。
In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation. References 1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. N Engl J Med. 2013 Dec 10;369(25):2379-90. 2. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. N Engl J Med. 2013 Dec 10;369(25):2391-405. 3. Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Blood. 2015 Dec 14;10.1182/blood-2015-11-681932. 4. Marty C, Pecquet C, Nivarthi H, Elkhoury M, Chachoua I, Tulliez M, et al. Blood. 2015 Nov 25;10.1182/blood-2015-11-679571. 5. Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C, et al. Leukemia. 2016 Feb 29;10.1038/leu.2016.47. Disclosures Constantinescu:Teva: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau.