携带TRAIL和Apoptin双基因溶瘤痘病毒对多发性骨髓瘤的杀伤作用及机制研究

Multiple myeloma(MM) is a B-cell malignancy that is currently felt to be incurable.Despite recently approved novel targeted treatments such as lenalidomide and bortezomib, most MM patients' relapse is emphasizing the need for effective and well-tolerated therapies for this deadly disease. One such therapy is oncolytic virotherapy, wherein viruses specifically infect and kill the malignant plasma cells,leaving normal cells intact.It also has no cross-resistance with chemotherapy.But most viruses only can be injected in tumor,recent studies have shown that oncolytic poxvirus can be systemic administration,and have anti-tumor effect of distant metastases.We have constructed a novel oncolytic poxvirus，carrying a green fluorescent protein, and found most MM cells can be infected, suggesting oncolytic poxvirus can play an important role in the treatment of MM.The genomes of tumor cells are invariably altered at multiple sites and several important signaling pathways are always disrupted at the same time; a combination of genes that target totally different aspects of tumor biology would achieve more potent inhibition in a wide variety of tumors.Both tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and Apoptin have strong anti-MM effect, particularly in the TRAIL-based combination therapy can successfully cleared MM stem cells.And they have the theoretical basis of synergistic antitumor effect.So,we use the gene linker, that is, sequence encoding the four amino acids IETD, to join the TRAIL gene and the Apoptin gene to form TRAIL-IETD-Apoptin and inserting it into oncolytic poxvirus vector OncoPoxV to construct OncoPoxV-TRAIL-IETD Apoptin.We will study the anti-myeloma of the novel oncolytic poxvirus, carrying TRAIL and Apoptin gene,in vivo and in vitro.And then we will study the anti-tumor mechanism on myeloma stem cell.

多发性骨髓瘤(MM)目前仍无法治愈，易耐药，疾病复发，迫切需要研究新的治疗措施。溶瘤病毒具有肿瘤靶向性且与化疗没有交叉耐药，但大多病毒只能局部使用，新近研究表明溶瘤痘病毒可静脉用药，具有全身抗肿瘤作用，且可携带治疗性基因达到病毒治疗和基因治疗联合的目的。我们已构建携带绿色荧光蛋白的溶瘤痘病毒，发现其能有效感染MM细胞，提示溶瘤痘病毒可在治疗MM中发挥重要作用。但肿瘤的发生往往是多基因的改变。单一的基因治疗或单一的靶点抑制无法根治肿瘤。因此我们提出双基因-病毒治疗MM。TRAIL和Apoptin，都有很强抗MM作用，特别是以TRAIL为基础的联合治疗能清除MM干细胞，且两者有协同作用的理论基础。因此我们应用连接工具IETD连接TRAIL和Apoptin，构建靶向MM的新型双基因溶瘤痘病毒，进一步研究新型双基因溶瘤痘病毒对骨髓瘤细胞及对骨髓瘤干细胞的杀伤作用及机制。

溶瘤病毒治疗是近年来发展非常迅速的一种恶性肿瘤治疗新方法，具有肿瘤靶向性且与化疗药物没有交叉耐药。溶瘤痘苗病毒作为病毒治疗的一种新型载体，可静脉用药，诱导抗肿瘤的免疫反应，具有全身抗肿瘤作用，且可携带治疗性基因达到病毒治疗和基因治疗联合的目的。本项目中，我们构建了携带miR-34a和Smac基因的新型溶瘤痘苗病毒VV-miR-34a和VV-Smac。 这两个新型溶瘤痘苗病毒均能有效的感染多发性骨髓瘤细胞株，增加靶基因的表达。miR-34a和Smac，都有很强的抗骨髓作用，且两者具有协同作用的理论基础；我们创新性将携带这两种基因的新型溶瘤痘病毒联合应用于骨髓瘤的治疗，即双基因-病毒治疗新方案。体外实验和动物实验证实了VV-miR-34a联合VV-Smac能协同杀伤多发性骨髓瘤细胞，显著延长小鼠的生存期。实验进一步证明了VV-miR-34a主要通过抑制凋亡抑制蛋白的表达以及通过下调Bcl-2，促进细胞色素c的释放来协同性增强VV-Smac诱导的细胞凋亡。综上，我们研究成果的创新性在于：我们首次利用溶瘤痘病毒作为载体增加miR-34a和Smac的表达，从而更加显著的杀伤骨髓瘤细胞，具有较好的临床应用前景，为溶瘤痘苗病毒治疗恶性血液肿瘤临床研究提供了理论与实验依据。