CN115418356A - 一种表达i型干扰素的hsv-1溶瘤病毒 - Google Patents
一种表达i型干扰素的hsv-1溶瘤病毒 Download PDFInfo
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Abstract
本发明公开了一种表达I型干扰素的HSV‑1溶瘤病毒,为以下任一种:表达鼠源IFN‑β的HSV‑1重组溶瘤病毒HSV‑1‑mIFN‑β‑C1、HSV‑1‑mIFN‑β‑C2、HSV‑1‑mIFN‑β‑C3,或者表达人源IFN‑β的HSV‑1重组溶瘤病毒HSV‑1‑hIFN‑β‑C1、HSV‑1‑hIFN‑β‑C2、HSV‑1‑hIFN‑β‑C3;所述HSV‑1‑mIFN‑β‑C1、HSV‑1‑hIFN‑β‑C1是将HSV‑1的一个γ34.5基因拷贝保留,将HSV‑1的另外一个γ34.5基因拷贝插入IFN‑β的病毒株;所述HSV‑1‑mIFN‑β‑C2、HSV‑1‑hIFN‑β‑C2的构建方法是将HSV‑1的一个γ34.5基因拷贝插入IFN‑β,将HSV‑1的另一个γ34.5移码突变后基因功能失活的病毒株;所述HSV‑1‑mIFN‑β‑C3、HSV‑hIFN‑β‑C3是将HSV‑1的两个γ34.5基因拷贝都插入IFN‑β。本发明以HSV‑1溶瘤病毒为载体,开发一种表达I型干扰素的肿瘤免疫治疗药物HSV‑1‑IFN‑β,HSV‑1‑IFN‑β可以利用病毒的感染特性将IFN‑β基因递送到肿瘤内部,在肿瘤细胞裂解的同时通过表达IFN‑β引起机体的抗肿瘤免疫应答,并极大地促进了HSV‑1溶瘤病毒在肿瘤细胞中的增殖,最终起到治疗肿瘤的目的。
Description
技术领域
本发明属于生物技术领域,具体涉及一种表达I型干扰素的HSV-1溶瘤病毒。
背景技术
I型干扰素(Interferon,IFN)是各种细胞(如成纤维细胞、白细胞、自然杀细胞等)对细菌、病毒、寄生虫等病原体以及癌症和其他外源细胞应激反应时分泌表达的一种天然蛋白质,其在先天免疫和适应性免疫的激活过程中都扮演着重要角色。近年来,越来越多的证据表明,I型干扰素可以同时作用于肿瘤细胞和免疫细胞,进而抑制肿瘤生长。其中,对于肿瘤细胞,I型干扰素不仅可以通过阻断细胞周期进程和诱导细胞凋亡通过抑制增殖、调节细胞凋亡、分化、迁移和细胞表面抗原表达发挥直接抗肿瘤作用,I型干扰素还可以刺激固有和适应性细胞毒性淋巴细胞群(T细胞、自然杀伤细胞NK、树突状细胞、固有淋巴样细胞ILCs),以及抑制抗肿瘤免疫的抑制性细胞(例如,骨髓源性抑制细胞MDSCs和调节性T细胞Treg),导致免疫细胞的激活,进而杀伤肿瘤。因此,肿瘤微环境中I型干扰素的释放是影响肿瘤生长和治疗的关键,激活或利用干扰素治疗肿瘤成为肿瘤免疫治疗的重要研究方向。
早在1986年,美国FDA首次批准I型干扰素rhIFNα-2a用于晚期黑色素瘤的治疗。这可以称之为第一代作用于I型干扰素通路的药物。之后的临床研究显示,I型干扰素通路的激活是刺激肿瘤抗原交叉呈递和肿瘤特异性CD8 T细胞激活的必需因素。在多种肿瘤模型中,通过修饰环核苷酸的瘤内传递,I型干扰素的治疗可以有效和持久的肿瘤消退。因此,I型干扰素在治疗癌症方面具有巨大潜能。目前Ⅰ型干扰素已在血液系统肿瘤(如毛细胞白血病、滤泡性淋巴瘤、慢性髓性白血病和多发性骨髓瘤)和实体瘤(如恶性黑色素瘤、AIDS相关性Kaposi氏肉瘤、肾癌、内分泌性胰腺肿瘤)的治疗中得到广泛应用。然而,全身性的I型干扰素免疫治疗的一个潜在的严重问题是“细胞因子风暴”的发生。免疫信号的持续激活会导致细胞因子的过量产生,导致严重中毒甚至死亡,这些副作用直接限制了I型干扰素的使用。因此,开发新一代的干扰素药物以更有效地靶向肿瘤组织、增强I型干扰素的半衰期以及有效减少系统性给药的副作用依然是该领域的重要研究方向。
HSV-1是一类有包膜的双链DNA病毒,属于α-疱疹病毒亚家族。目前,HSV-1已被广泛应用于重组溶瘤病毒治疗药物的开发研究,同时也是首个被美国FDA批准用于抗癌治疗的溶瘤病毒。相较于其他类型的溶瘤病毒,HSV-1拥有更长的基因组(约150kb),能接纳更多外源基因的插入,因此HSV-1被广泛用作针对肿瘤基因治疗的基因递送平台。其中,RichardG.Vile课题组已经将I型干扰素IFN-β整合到病毒或者溶瘤病毒载体内用于小鼠肝癌的治疗,并且在大鼠和恒河猴中证实了其安全性。然而,由于IFN-β能有效激活抗病毒免疫反应,将I型干扰素整合到病毒体内仍将面临巨大挑战。因此,如何解决IFN-β在溶瘤病毒治疗过程中激活抗肿瘤免疫和抗病毒免疫间的平衡是需要攻克的难题。
发明内容
本发明的目的在于提供一种表达I型干扰素的HSV-1溶瘤病毒。
本发明的技术思路在于I型干扰素可以同时作用于肿瘤细胞和免疫细胞,进而抑制肿瘤生长,在治疗癌症方面具有巨大潜能。然而,全身性的I型干扰素免疫治疗的一个潜在严重问题是“细胞因子风暴”的发生。免疫信号的持续激活会导致细胞因子的过量产生,导致严重中毒甚至死亡,这些副作用直接限制了I型干扰素的使用。HSV-1溶瘤病毒作为针对肿瘤基因治疗的基因递送平台,通过局部注射可以有效解决I型干扰素全身性的副作用。
然而,I型干扰素能够激活机体产生抗HSV-1病毒免疫,导致HSV-1溶瘤病毒在肿瘤细胞中的增殖受到抑制,且这一过程依赖于宿主细胞内双链RNA依赖的蛋白质激酶(Double-stranded RNA-dependent protein kinase,PKR)的激活。激活后的PKR导致真核细胞翻译起始因子eIF2α发生磷酸化,从而终止宿主细胞的蛋白质合成,最终使病毒的合成受抑制,进而发挥抗病毒的作用。因此,抑制PKR通路可能有效增强溶瘤病毒的增殖。与此同时,研究发现HSV-1的神经毒性因子γ34.5(ICP34.5)可以有效抑制PKR的激活,使病毒的复制增殖能以继续进行。
然而,由于γ34.5基因表达神经毒性因子ICP34.5,因此,通常情况下制备HSV-1溶瘤病毒要敲除γ34.5基因。由于HSV-1中存在两个γ34.5基因拷贝,因此,我们在HSV-1中整合IFN-β的同时保留一个拷贝的γ34.5基因,既可以提升溶瘤病毒治疗的有效性,又可以克服系统使用I型干扰素带来非靶向性细胞毒性的局限,还可以极大的促进HSV-1溶瘤病毒在肿瘤细胞中的增殖,最终实现I型干扰素激活抗肿瘤免疫的同时避免激活抗病毒免疫,进而增强抗病毒效果。
为了实现上述目的,本发明采用的技术方案如下:
一种表达I型干扰素的HSV-1溶瘤病毒,包括三株表达鼠源IFN-β的HSV-1重组溶瘤病毒HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2、HSV-1-mIFN-β-C3,三株表达人源IFN-β的HSV-1重组溶瘤病毒HSV-1-hIFN-β-C1、HSV-1-hIFN-β-C2、HSV-1-hIFN-β-C3;
其中,所述HSV-1-mIFN-β-C1、HSV-1-hIFN-β-C1的构建方法是将HSV-1的一个γ34.5基因拷贝保留,将HSV-1的另外一个γ34.5基因拷贝插入IFN-β的病毒株;
所述HSV-1-mIFN-β-C2、HSV-1-hIFN-β-C2的构建方法是将HSV-1的一个γ34.5基因拷贝插入IFN-β,将HSV-1的另一个γ34.5移码突变后基因功能失活的病毒株;
所述HSV-1-mIFN-β-C3、HSV-IFN-β-C3的构建方法是将HSV-1的两个γ34.5基因拷贝都插入IFN-β。
本发明的有益效果在于:本发明利用HSV-1溶瘤病毒为载体,开发一种表达I型干扰素的肿瘤免疫治疗药物HSV-1-IFN-β(HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2、HSV-1-mIFN-β-C3、HSV-1-hIFN-β-C1、HSV-1-hIFN-β-C2、HSV-1-hIFN-β-C3)。HSV-1-IFN-β溶瘤病毒旨在利用病毒的感染特性将IFN-β基因递送到肿瘤内部,在肿瘤细胞裂解的同时通过表达IFN-β引起机体的抗肿瘤免疫应答,最终起到治疗肿瘤的目的。其中,HSV-1-mIFN-β-C1和HSV-1-hIFN-β-C1溶瘤病毒在激活强效的抗肿瘤免疫的同时极大地促进HSV-1溶瘤病毒在肿瘤中的增殖。本发明数据显示,HSV-1-IFN-β溶瘤病毒治疗可以有效抑制肿瘤生长,改变肿瘤微环境,在小鼠肿瘤模型中形成免疫记忆和人类APCs的强大激活。本发明为肿瘤治疗提供了一种新的策略。
附图说明
图1为构建HSV-IFN-β重组溶瘤病毒的过程。其中,A为重组溶瘤病毒HSV-1-IFN-β的构建策略图,表达IFN-β单/双拷贝基因的HSV-1重组溶瘤病毒的测序验证;B为将pX459-γ34.5-KO重组质粒和纯化后的同源重组片段共同转染293T细胞,同时加入野生型HSV-1-KOS病毒,转染后36h在荧光显微镜下的镜检图;在激发光下可见293T细胞内有绿色荧光蛋白表达,表明同源臂片段转染成功;C为收集图B中细胞上清,感染VERO细胞,在荧光显微镜下挑取荧光病毒噬斑,显微镜下的镜检图,结果表明,随着逐轮噬斑挑选,病毒的纯度不断提高,最终,经四轮挑选后,所有被病毒感染的细胞均带有绿色荧光;D为11株溶瘤病毒基因组扩增产物的琼脂糖凝胶验证,第1泳道为DNA marker,第2泳道为野生型HSV-1-KOS基因组的扩增产物,第3-13泳道是分离到的11株溶瘤病毒基因组的扩增产物;E为HSV-1-IFN-β的测序结果。
图2为HSV-1-IFN-β重组溶瘤病毒在感染的肝癌细胞中表达IFN-β的结果。其中,A为分别用HSV-1-△γ34.5、HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2和HSV-1-mIFN-β-C3溶瘤病毒感染Hepa1-6细胞,24h后提取病毒感染的细胞RNA,反转录产物用m-IFN-β引物进行qPCR验证(****P<0.0001,student’st-test)。B为用HSV-1-mIFN-β-C1溶瘤病毒感染Hepa1-6细胞,24h后提取病毒感染的细胞RNA,反转录产物用m-IFN-β引物进行qPCR验证。C为用HSV-1-mIFN-β-C1溶瘤病毒感染Hepa1-6细胞,分别在第0、12、24、36h收取的细胞,提取RNA,反转录产物用m-IFN-β引物进行qPCR验证。D为用HSV-1-mIFN-β-C1溶瘤病毒感染Hepa1-6细胞,24h后提取病毒感染的细胞RNA,反转录产物用m-IFIT1引物进行qPCR验证。
图3为HSV-1-mIFN-β重组溶瘤病毒有效抑制小鼠肝肿瘤。每隔3日进行一次治疗和肿瘤体积测量,总共进行6次治疗。其中,A为HSV-1-mIFN-β-C1溶瘤病毒治疗期间小鼠肿瘤体积的变化趋势。每隔3天进行一次治疗和测量,共进行6次治疗,黑色箭头代表最后一次治疗结束。误差线代表平均±SEM(n=5)。(*p<0.05,**p<0.01,***p<0.001,****p<0.0001,one-way ANOVA),B为HSV-1-mIFN-β-C1溶瘤病毒治疗期间小鼠体重的变化趋势,C为最后一次重组溶瘤病毒治疗结束后3天,将小鼠背部肿瘤切除后进行拍照记录。
图4为HSV-1溶瘤病毒治疗后的肿瘤组织的免疫荧光染色。其中,A为最后一次治疗后第3天将小鼠处死,用CD4和CD8抗体对切除的肿瘤组织进行免疫荧光染色,荧光显微镜下观察阳性细胞的分布情况。比例尺:100μm,B为对来自3组不同肿瘤组织样本的CD4免疫荧光图中的阳性细胞进行量化分析。误差线代表平均±SEM(n=3)。C为对来自3组不同肿瘤组织样本的CD8免疫荧光图中的阳性细胞进行量化分析。误差线代表平均±SEM(n=3)。
具体实施方式
以下结合附图和具体实施方式对本发明作进一步详细说明。
1、构建携带I型干扰素IFN-β的HSV-1-IFN-β型溶瘤病毒
为了探究γ34.5缺失对IFN-β诱导的抗病毒免疫的影响,我们利用CRISPR/Cas9基因编辑技术,构建了三株表达鼠源IFN-β的HSV-1重组溶瘤病毒HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2和HSV-1-mIFN-β-C3,三株表达人源IFN-β的HSV-1重组溶瘤病毒HSV-1-hIFN-β-C1、HSV-1-hIFN-β-C2和HSV-1-hIFN-β-C3。本实验在IFN-β细基因片段两端分别加上病毒γ34.5基因的同源片段(上下游各500bp)。此外,为了方便后续分离成功整合上外源基因的重组溶瘤病毒,本实验在细胞因子基因的后端连接上由CMV启动子启动的绿色荧光蛋白基因,通过荧光筛选进行后续的病毒分离和纯化,构建重组溶瘤病毒。其中,HSV-1-mIFN-β-C1和HSV-1-hIFN-β-C1保留一个γ34.5基因,另外一个γ34.5基因插入m-IFN-β的病毒株;HSV-1-mIFN-β-C2和HSV-1-hIFN-β-C2的一个γ34.5基因插入m-IFN-β,另一个γ34.5移码突变后基因功能失活的病毒株;HSV-1-mIFN-β-C3和HSV-1-hIFN-β-C3则是两个拷贝都插入了IFN-β(图1A)。具体为:①在0.2mL EP管中依次添加酶切组分3ul BBSI酶,3ul Buffer,5ug质粒,补足ddH2O至30ul,旋涡振荡混匀,瞬时离心后,置于37℃水浴锅,反应30min。酶切反应结束后,配置浓度为1%的琼脂糖凝胶,取1μL酶切产物上样,同时以未酶切的质粒作对照,进行琼脂糖凝胶电泳,验证质粒酶切是否成功。若酶切未完全,则适当延长酶切时间,待质粒被完全酶切后,65℃加热10min使酶彻底失活。酶切产物立即进行过柱纯化。②接着在1.5mL EP管内依次添加1μl pX459酶切产物(50ng/μl),1μl sgRNA退火产物,1μl T4 DNAligase,2μl 10×T4 DNA ligase Buffer,15μl ddH2O,吹打混匀,短暂离心,然后置于4℃进行过夜连接。③之后将重组质粒转化到DH5α大肠杆菌并于37℃恒温培养箱内倒置过夜培养。④将pX459-γ34.5-KO重组质粒和纯化的同源臂片段共同转染293T细胞,同时加入野生型HSV-1-KOS病毒,转染后36h,观察细胞中是否有荧光蛋白表达。⑤在同源臂上设计引物扩增插入片段,通过条带大小和测序结果验证重组病毒。
通过上述构建过程,结果显示,293T细胞内有绿色荧光蛋白表达,表明同源臂片段转染成功(图1B)。随后收集上述细胞上清,并感染VERO细胞,此为第一轮病毒纯化,在荧光显微镜下可以看到明显的荧光病毒噬斑(图1C)。为了得到更纯的重组HSV-IFN-β病毒,我们经四轮噬斑挑选,最终所有被病毒感染的细胞均带有绿色荧光(图1C)。经过4轮荧光病毒噬斑的挑选,共获得11株病毒,将其感染细胞,PCR扩增HSV-1基因组的ICP34.5片段,结果显示我们得到了多种不同的重组HSV-1溶瘤病毒(图1D)。PCR产物跑胶图显示第3、4、5、13泳道同时扩增出一个插入IFN-β的长条带,和未插入的短扩增条带。接着对HSV-1-mIFN-β的扩增产物进行TA克隆,测序验证结果显示第3、4泳道的短片段扩增产物与原始γ34.5基因序列一致,分别为HSV-1-mIFN-β-C1、HSV-1-hIFN-β-C1;第5、13泳道的短片段扩增产物均不能与原始γ34.5基因序列匹配,因此,第5、13泳道分别为HSV-1-mIFN-β-C2、HSV-1-hIFN-β-C2(图1E)。此外,第6、7、8泳道只扩增出出一个插入IFN-β的长条带,表明其是两个ICP34.5的位置都插入了IFN-β基因,其中6、7泳道分别为HSV-1-mIFN-β-C3、HSV-1-hIFN-β-C3。
2、HSV-1-IFN-β重组溶瘤病毒在感染的癌细胞中高表达IFN-β
人和鼠的I型干扰素IFN-β在生物体内有同样的功能,但结构不同,因此,人源的干扰素在小鼠体内无法发挥功能。本实施例中,我们以鼠源干扰素为例,在小鼠肝癌细胞中进行研究。其中,HSV-1-KOS为野生型HSV-1病毒株,HSV-1-Δγ34.5为缺失γ34.5的HSV-1病毒株。首先,我们分别用HSV-1-Δγ34.5、HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2和HSV-1-mIFN-β-C3感染Hepa1-6细胞,分析mIFN-β的表达情况。结果表明,HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2和HSV-1-mIFN-β-C3感染后,细胞都能显著表达I型干扰素IFN-β,且HSV-1-mIFN-β-C1表达水平更高(图2A)。因此我们以HSV-1-mIFN-β-C1为例,研究其在细胞水平上表达I型干扰素IFN-β的能力。首先,我们分别用HSV-1-KOS、HSV-1-Δγ34.5和HSV-1-mIFN-β-C1感染Hepa1-6细胞,分析mIFN-β的表达情况。结果表明,HSV-1-mIFN-β-C1感染的Hepa1-6细胞中可检测到大量mIFN-β的mRNA,而野生HSV-1-KOS和HSV-1-Δγ34.5感染细胞中的m-IFN-β的RNA水平都与对照组(未添加病毒感染)无显著性差异(图2B)。此外,HSV-1-mIFN-β-C1感染Hepa1-6细胞后,m-IFN-β的mRNA在24h表达量最高(图2C)。同时我们检测了IFN-β通路下游的ISG,结果显示HSV-1-mIFN-β能有效刺激干扰素刺激基因IFIT1的转录(图2D)。由此可以证明,表达I型干扰素的HSV-1重组溶瘤病毒在感染的肝癌细胞中高表达IFN-β,且HSV-1-mIFN-β-C1诱导肿瘤细胞表达I型干扰素的能力最强。
3、HSV-1-IFN-β重组溶瘤病毒有效抑制小鼠肝肿瘤
本实施例中,我们以HSV-1-mIFN-β-C1溶瘤病毒为例,在小鼠肝癌模型中进行研究。以敲除γ34.5基因的HSV-Δγ34.5为对照组,将HSV-mIFN-β重组溶瘤病毒直接注射至小鼠肝肿瘤内,结果显示,小鼠肿瘤的生长受到不同程度的抑制,与HSV-Δγ34.5病毒相比,HSV-mIFN-β-C1溶瘤病毒对肿瘤生长的抑制作用更显著(图3A)。对治疗过程中的小鼠体重进行监测,结果表明溶瘤病毒治疗对小鼠的体重变化无显著性影响,表明了HSV-mIFN-β-C1溶瘤病毒治疗肿瘤过程中基本的安全性(图3B)。同样,将最后一次治疗后的肿瘤组织从小鼠背部剥离,拍照并称重,结果显示HSV-mIFN-β治疗6次后,小鼠肿瘤大小与对照组相比也具有显著性差异(图3C)。
4、HSV-1-IFN-β治疗增强小鼠肿瘤内CD4+和CD8+T细胞的浸润
本实施例中,我们以鼠源干扰素为例,在小鼠肝癌模型中进行研究。将治疗后的肿瘤组织进行免疫荧光染色,检测肿瘤内部CD4+和CD8+T细胞的浸润情况,结果显示,相较于未添加HSV-1病毒组,注射溶瘤病毒HSV-Δγ34.5能够促进CD4+、CD8+T细胞向小鼠肿瘤内部的浸润,且HSV-1-mIFN-β治疗后的小鼠肿瘤组织中的CD4+、CD8+T细胞浸润程度更高(图4A)。用Image J对显微镜下的阳性细胞进行量化分析,结果显示,表明溶瘤病毒能够促进CD4+、CD8+T细胞对肿瘤组织的浸润,且整合上细胞因子的重组溶瘤病毒对免疫细胞向肿瘤内部的浸润有着更显著的促进作用,这与小鼠肝肿瘤模型治疗疗效的结果一致(图4B-C)。
Claims (2)
1.一种表达I型干扰素的HSV-1溶瘤病毒,其特征在于,其为以下任一种:
表达鼠源IFN-β的HSV-1重组溶瘤病毒HSV-1-mIFN-β-C1、HSV-1-mIFN-β-C2、HSV-1-mIFN-β-C3,或者表达人源IFN-β的HSV-1重组溶瘤病毒HSV-1-hIFN-β-C1、HSV-1-hIFN-β-C2、HSV-1-hIFN-β-C3;
其中,所述HSV-1-mIFN-β-C1、HSV-1-hIFN-β-C1的构建方法是将HSV-1的一个γ34.5基因拷贝保留,将HSV-1的另外一个γ34.5基因拷贝插入IFN-β的病毒株;
所述HSV-1-mIFN-β-C2、HSV-1-hIFN-β-C2的构建方法是将HSV-1的一个γ34.5基因拷贝插入IFN-β,将HSV-1的另一个γ34.5移码突变后基因功能失活的病毒株;
所述HSV-1-mIFN-β-C3、HSV-hIFN-β-C3的构建方法是将HSV-1的两个γ34.5基因拷贝都插入IFN-β。
2.如权利要求1所述的一种表达I型干扰素的HSV-1溶瘤病毒在制备治疗肿瘤的药物中的应用。
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