Thymidine Kinase-Deleted, let7a-Regulated Vaccinia Virus Specifically Infects and Lyses Myeloma Cells in a Mouse Myeloma Model
Thymidine Kinase-Deleted, let7a-Regulated Vaccinia Virus Specifically Infects and Lyses Myeloma Cells in a Mouse Myeloma Model
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DOI：
10.1182/blood.v124.21.2082.2082
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发表时间：
2014-12
期刊：
影响因子：
20.3
通讯作者：
Muneyoshi Futami;Kota Sato;Takafumi Nakamura;A. Tojo
中科院分区：
文献类型：
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作者：
Muneyoshi Futami;Kota Sato;Takafumi Nakamura;A. Tojo
背景：牛痘病毒是一种感染和溶解癌细胞的新型癌症治疗工具。针对实体瘤的临床试验目前正在进行中，并报道了令人鼓舞的结果。尽管体外研究表明血液系统恶性肿瘤一般只有适度的易感性，但我们发现多发性骨髓瘤对牛痘病毒的易感性异常高，因此，我们将重点放在骨髓瘤上。由于肿瘤特异性感染是成功溶瘤治疗的关键，我们修改了牛痘基因组中的胸苷激酶（TK）基因和B5R基因，使其更具肿瘤特异性。通过体外和体内小鼠感染模型验证其有效性和安全性。方法：痘苗TK是病毒复制所必需的，但在肿瘤细胞中是不可缺少的，因为肿瘤产生的高水平TK可以替代它。为了删除TK基因，我们构建了具有TK同源序列的质粒载体（pTK），并将具有PSFL110启动子的萤光素酶（Fluc）基因插入到TK基因的中间。通过将pTK-Fluc和亲本牛痘病毒LC16mO（来自Lister克隆16）共转染143B细胞，发生同源重组，产生tk缺失的牛痘病毒。痘苗B5R基因是病毒从感染细胞向邻近细胞传播所必需的。为了调节B5R基因的表达，抑制病毒在正常细胞中的传播，我们在pB5R质粒的B5R下游插入了let-7a靶序列。Let-7a是一种在肿瘤细胞中缺失表达但在大多数正常细胞中保持表达的微RNA，因此我们假设正常细胞中内源性Let-7a会抑制B5R，抑制病毒传播，提高安全性。通过将pB5Rgfp-let7a和tk缺失的牛痘病毒共转染RK13细胞，发生同源重组，生成tk缺失的let-7a调控的牛痘病毒。结果：我们首先感染了不同的细胞系，包括Kasumi-1、NB4、NALM6、Jurkat、OM9；22, K562（这些来自白血病），FL218, Daudi（淋巴瘤），MT2, TLO-m1 (ATL), RPMI8226, U266, NCI-H929（骨髓瘤）与牛痘病毒。非常有趣的是，骨髓瘤来源的细胞系表现出异常高的易感性（比其他细胞系高10-100倍），这种易感性与实体瘤一样高；因此我们选择多发性骨髓瘤作为研究对象。为了证实let-7a对B5R的调控能改善骨髓瘤特异性感染，我们用重组B5Rgfp-let7a痘苗病毒感染了骨髓瘤细胞系RPMI8226和正常皮肤成纤维细胞。体外感染RPMI8226细胞，即使滴度很低（MOI=0.1），也显示出显著的感染性，而对照正常细胞（人皮肤成纤维细胞）完全不受感染。在没有let7a调控的情况下，牛痘病毒以相同滴度感染骨髓瘤细胞和正常细胞，这表明let-7a介导的调控显然有效。将1x107 RPM8226-Rluc细胞皮下注射免疫缺陷小鼠（CB.17-SCID）作为体内感染模型。4周后，静脉注射1 × 106 pfu病毒（Parent; TK-deleted； TK和let7a双调控），每周用肾和萤火虫荧光素酶体内显像系统检测肿瘤体积和病毒量。在没有任何调控的情况下，牛痘病毒不仅会感染骨髓瘤细胞，还会感染正常组织，小鼠的耳、鼻、口、足、尾出现麻袋病变，在21 ~ 24天内死亡。tk缺失显著减轻了病毒毒性，但仍导致感染后42天死亡。相比之下，TK和let-7a双调控牛痘病毒感染在骨髓瘤中明显受到限制，所有小鼠都存活，骨髓瘤的大小不断缩小。结论：tk -let7a双调控牛痘病毒可特异性感染和杀伤骨髓瘤细胞，具有临床应用价值。无相关利益冲突需要申报。
Background: Vaccinia virus is a tool for a novel cancer therapy that infects and lyses cancer cells. Clinical trials targeting solid tumors are currently ongoing, and promising results are reported. Although in vitro studies suggest that hematological malignancies have only modest susceptibility in general, we found that multiple myeloma shows an exceptionally high susceptibility to vaccinia virus, therefore, we focused on myeloma. Because tumor specific infection is the key for a successful oncolytic therapy, we modified thymidine kinase (TK) gene and B5R gene in the vaccinia genome to make it more tumor-specific. Efficacy and safety was determined by the in vitro and in vivo mouse infection models. Methods: Vaccinia TK is required for viral replication, but it is dispensable in tumor cells because high level of TK produced by the tumor can substitute it. In order to delete TK gene, we generated a plasmid vector which have a homologous sequence of TK (pTK), and the firefly luciferase (Fluc) gene with the PSFL110 promoter was inserted into the middle of TK gene. By the co-transfection of 143B cells with the pTK-Fluc and parental vaccinia virus LC16mO (derived from Lister clone 16), a homologous recombination occurred and the TK-deleted vaccinia virus was generated. Vaccinia B5R gene is essential for the viral transmission from infected cells to adjacent cells. To regulate B5R gene expression and inhibit viral transmission in normal cells, we inserted a let-7a target sequence into the downstream of B5R in the pB5R plasmid. Let-7a is a micro RNA of which expression is lost in tumor cells but maintained in most of the normal cells, therefore, we hypothesized that endogenous let-7a in normal cells will inhibit B5R, inhibit viral transmission, and improve safety. By the co-transfection of RK13 cells with the pB5Rgfp-let7a and TK-deleted vaccinia virus, a homologous recombination occurred, and TK-deleted let-7a-regulated vaccinia virus was generated. Results: To find out a good target for this therapy, we first infected different cell lines including Kasumi-1, NB4, NALM6, Jurkat, OM9;22, K562 (these are from leukemia), FL218, Daudi (lymphoma), MT2, TLO-m1 (ATL), and RPMI8226, U266, NCI-H929 (myeloma) with a vaccinia virus. Very interestingly, myeloma-derived cell lines showed an exceptionally high susceptibility (10-100 times higher than others), and this susceptibility was as high as solid tumors; therefore we chose multiple myeloma as the target. To confirm that regulation of B5R by the let-7a improves myeloma-specific infection, we infected a myeloma cell line RPMI8226 as well as normal skin fibroblasts with the recombinant B5Rgfp-let7a vaccinia virus. In vitro infection of a RPMI8226 cells showed a significant infectivity even with a very low titer (MOI=0.1), while control normal cells (human skin fibroblasts) were not infected at all. Without the let7a-regulation, the vaccinia virus infected both myeloma cells and normal cells at the same titer, suggesting that let-7a-mediated regulation clearly works. As an in vivo infection model, 1x107 RPM8226-Rluc cells were injected into immunodeficient mice (CB.17-SCID) subcutaneously. Four weeks later, 1x106 pfu of virus (Parent; TK-deleted; TK and let7a double regulated) was administered via an intravenous injection, and tumor volume and the amount of virus were determined weekly by the in vivo imaging system with renilla and firefly luciferases. Without any regulation, vaccinia virus infected not only myeloma cells but also normal tissues, and mice developed pock lesions in the ear, nose, mouth, foot, and tail, resulting in death within 21 to 24 days. TK-deletion significantly alleviated viral toxicity but still caused death 42 days after infection. In contrast, infection with TK and let-7a double regulated vaccinia virus was clearly limited in myeloma, and all mice remained alive and the size of myeloma shrunk continuously. Conclusions: These data suggest that the TK-let7a-double regulated vaccinia virus infects and kills myeloma cells specifically, and will be a good candidate for the future clinical application. Disclosures No relevant conflicts of interest to declare.