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通讯作者:

王祎(1983-),男,四川南充人,硕士生导师,主要从事肝脏疾病的基因治疗研究。E-mail:wy4504228@163.com

中图分类号:Q812R

文献标识码:A

文章编号:2096-8965(2021)01-0083-06

DOI:10.12287/j.issn.2096-8965.20210112

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目录contents

    摘要

    腺病毒载体是目前应用最为广泛的基因治疗病毒载体之一。相较其他病毒载体,腺病毒具有较高的基因转导效率,广泛的组织亲和性,不整合入宿主基因组等优点。腺病毒可以被改造为复制缺陷病毒载体和选择性复制的溶瘤病毒载体。腺病毒载体较强的免疫原性使其可以被应用于基因疫苗和抗癌治疗中。目前众多的临床试验都证实了腺病毒载体在上述治疗试验中的安全性和有效性。

    Abstract

    Adenovirus vector is one of the most widely used viral vectors for gene therapy. Compared with other viral vectors, adenovirus has various advantages such as high transduction effi ciency, broad tropism for diff erent tissues and epichromosomal persistence in the host cell, etc. Adenovirus vectors can be modifi ed as replication-deficient and replication-selective oncolytic virus vector. The strong immunogenicity of adenovirus vector makes it suitable to be applied in genetic vaccines and anti-cancer therapies. At present, many clinical trials have confirmed the safety and effectiveness of adenovirus vector in the above-mentioned therapeutic attempts.

  • 1 腺病毒载体的生物学特性

  • 腺病毒(Adenovirus, Ad)是一种自然界普遍存在的非包膜DNA病毒,可感染人类。腺病毒由一个二十面体蛋白衣壳容纳26~45kb的线性双链DNA基因组所构成。腺病毒衣壳直径90~100nm,由六邻体(Hexon)、五邻体基底(Penton Base)、纤突(Fiber)、衣壳蛋白前体p Ⅲ a、p Ⅵ、p Ⅷ、衣壳蛋白Ⅸ和病毒核心蛋白(Ⅴ、Ⅶ和Ⅹ)等结构蛋白组成[1]。到目前为止,已有7个亚群(Subgroup A-G), 80余种人腺病毒基因型被鉴定(http://hadvwg.gmu.edu)。对人5型腺病毒(HAd5, C亚群)感染途径的研究发现,病毒感染依赖于宿主细胞表面的柯萨奇病毒-腺病毒受体(Coxsackievirus-adenovirus Receptor,CAR)与病毒衣壳上纤突蛋白远端结构域的相互作用 [2]。此外,如CD46、DSG2和唾液酸受体 [3-5] 等其他受体参与B或D亚群腺病毒对宿主细胞的感染。在随后的病毒入胞过程中,宿主细胞表面的整合素 αV也会扮演重要作用 [6]。病毒DNA通过宿主细胞核膜孔复合体进入细胞核,但不整合入宿主细胞基因组。由于过往的腺病毒感染史,大多数人携带一种或多种人腺病毒血清型的中和抗体(Neutralizing Antibodies,NAbs)[7]

  • 2 用于基因治疗的腺病毒载体类型

  • 自腺病毒载体被发明应用于基因治疗以来,人们为提高其基因容量,感染效率,基因转导持续时间和安全性,对腺病毒载体进行了持续地改造。第一代腺病毒载体是将E1A和E1B基因删除构建而成的复制缺陷病毒,可以搭载约4.5kb的外源基因 [8]。生产该类病毒时需要使用稳定表达E1A和E1B基因的细胞系 [9]。非必需基因E3的敲除使得E1/E3双敲除病毒拥有更大的转基因空间。为克服第一代载体引起宿主强烈的免疫应答的缺点,研究人员进一步删除了其他早期基因并扩展转基因空间至10.5kb。这些载体包括E2A删除的温度敏感载体 [10],E2B删除 [11] 以及E4删除的载体 [12]。第二代病毒载体的晚期基因表达和宿主细胞毒性T淋巴细胞反应明显降低,体内的基因转导时限显著延长。然而,E2和/或E4基因的缺失会导致生产病毒滴度降低 [13]。第三代腺病毒载体的基因组只保留了ITRs和包装信号序列,因此可容纳约36kb的外源基因,被称为“高容量”腺病毒载体(High Capacity Ads, HCAds)[14]。由于删除了大部分必需的病毒基因,在生产过程中需要引入表达腺病毒装配所需的基因产物的辅助病毒帮助其组装。为避免辅助病毒对载体病毒的污染,辅助病毒自身包装信号序列两侧插入了loxP位点。在可稳定表达Cre酶的病毒生产细胞系中,辅助病毒基因组因Cre对Loxp位点的切割无法进行组装 [15]。相较早期的腺病毒载体,HCAds的免疫原性大大降低,在宿主细胞内的转导时间更长。且明显增大的转基因容量可使其容纳多个转基因元件,或者更大的天然启动子和增强子来模拟治疗基因的生理表达水平。

  • 另一类腺病毒载体被称为选择性复制的腺病毒载体(Conditionally Replicating Adenovirus, CRAds)。如前所述,腺病毒E1B基因产物通过结合和灭活宿主表达的p53蛋白促进病毒在正常细胞内扩增。而大多数肿瘤细胞中的p53基因处于失活状态,可容许E1B缺失的腺病毒扩增。研究者们据此研发出可以选择性地在肿瘤细胞中扩增的CRAds。最初的CRAds载体是将E1B序列部分删除(E1B-55K)[16],选择性扩增的CRAds最终会通过溶瘤作用杀死肿瘤细胞,释放新生病毒来破坏剩余的肿瘤细胞。新一代的CRAds则是通过删除E1A蛋白中CR2结构域的24个氨基酸而产生AdΔ24载体 [17]。CR2可与视网膜母细胞瘤蛋白(Retinoblastoma Protein, pRb)结合以释放病毒在正常细胞复制所需的S期激活转录因子(S-phase-activating Transcription Factor, E2F),由于癌细胞通常表达过量的E2F,删除CR2结构域的AdΔ24无须结合pRb便能获得足够的E2F,因此在各类肿瘤细胞中均表现出较高的复制能力和选择性 [18]

  • 3 腺病毒载体在基因治疗中的应用

  • 90 年代初,人们在大鼠肝细胞和肺组织中利用腺病毒载体表达 α1-抗胰蛋白酶(Alpha-1 Antitrypsin, A1AT)基因 [19]。随后,腺病毒载体被迅速用于各类单基因遗传疾病的临床治疗试验中。然而1999 年一位叫Jesse Gelsinger的患者在进行腺病毒载体搭载的鸟氨酸转氨酶(Ornithinetranscarbamylase, OTC)基因治疗试验中突然死亡,引起人们对基因治疗安全性的广泛关注 [20],使得腺病毒载体的基因治疗研究曾一度陷入停滞。研究者也意识到正因为腺病毒载体具有较强的免疫原性,使其可以被应用于治疗一些不受免疫反应影响,甚至依赖免疫反应的治疗过程 [1]。目前,基于腺病毒载体的基因治疗临床试验数占全球基因治疗试验总数的近一半,而主要的应用方向就是基因疫苗的研制和抗癌治疗。

  • 3.1 腺病毒载体作为基因疫苗

  • 目前,基于腺病毒载体的埃博拉(Ebola Virus Diease)疫苗在临床受试者中已经表现出特异性抗体和T细胞反应 [21, 22]。与此同时,联合表达主要流感病毒抗原(如HA、NP和M2)的腺病毒流感疫苗已经在包括预防H1N1和H5N1流感株的临床试验中进行了测试 [23, 24]。腺病毒HIV疫苗也已经处于大规模开发和测试阶段 [25, 26]。近年,中国研究团队研发了基于HAd5载体表达SARS-CoV-2刺突糖蛋白为抗原的腺病毒新冠疫苗正在进行临床试验。数据显示,大多数受试者在接种疫苗14天后迅速获得抗体和反应性T细胞,没有严重的不良反应 [27]。由欧洲研究团队开发的基于黑猩猩腺病毒载体ChAdOx1的SARS-CoV-2疫苗也正在进行大规模临床试验。初步数据表明,ChAdOx1-nCoV-19治疗可诱导抗SARS-CoV-2的体液和细胞免疫反应 [28]

  • 同时,重组腺病毒载体可以通过表达肿瘤相关抗原(Tumor-associated Antigens, TAA)和/或促进溶瘤过程来制造各类癌症疫苗。目前用于制造癌症疫苗的抗原包括前列腺癌抗原PSA,实体瘤抗原MAGE19A3,HPV相关肿瘤抗原HPV E6/E7,以及结直肠癌和胰腺癌抗原CEA等 [29]

  • 3.2 腺病毒载体在抗肿瘤治疗中的应用

  • 由于许多肿瘤细胞的p53抑癌通路都出现了紊乱,腺病毒载体可以被用来重新表达p53,以引起这些肿瘤细胞生长停滞或凋亡 [30]。2003年,表达抑癌基因p53的腺病毒载体今又生(Gendicine) 在中国获得批准,成为世界上第一种商业化的基因治疗药物 [31]。选择性复制的腺病毒载体可以通过溶瘤作用特异地杀死肿瘤细胞, ONYX-105(dl1520)是第一个进入临床试验的溶瘤病毒载体 [32]。随后,类似的病毒载体H101(Oncorine) 也在中国获得了临床使用批准 [33]

  • 3.2.1 腺病毒载体递送细胞“自杀基因(Suicide Gene)”

  • 除了表达抑癌基因p53,腺病毒载体还可以通过表达某些前体药物的转化酶来实现肿瘤细胞杀伤。例如,嘌呤核苷磷酸化酶(Purine Nucleoside Phosphorylase, PNP)将前体药物氟达拉滨单磷酸盐(F-ara-AMP)转化为对增殖细胞具有细胞毒性的氟腺嘌呤(Fluoroadenine)。由编码大肠杆菌PNP的腺病毒载体(Ad/PNP)合并静脉注射F-araAMP被纳入治疗晚期肿瘤患者的临床试验 [34]。又如单纯疱疹病毒胸苷激酶(Herpes Simplex VirusThymidine Kinase, HSV-TK)能将前体药物更昔洛韦(Ganciclovir, GCV)转化为对分裂细胞有毒性的核苷酸。根据这一原理,腺病毒-TK载体加更昔洛韦疗法被应用于多个治疗实体肿瘤的临床试验中 [35]。再比如胞嘧啶脱氨酶(Cytosine Deaminase, CD)能将前体药5-氟胞嘧啶(5-FC)转化为毒性5-氟尿苷(5-FU),后者在细胞内进一步加工成导致细胞胸苷酸合成受阻和DNA损伤的三磷酸5-氟尿嘧啶核苷(5-FUTP)和一磷酸脱氧核糖5-氟尿嘧啶核苷(5-FdUMP)。临床试验表明搭载CD和HSV-TK嵌合基因的HAd5-CD/TKrep载体与5-FC和更昔洛韦联合应用时,显示出长期有效性 [36]。另一种利用HAd5递送表达酵母CD酶与TK酶融合蛋白以及腺病毒死亡蛋白(Adenovirus Death Protein, ADP)的治疗方式HAd5-yCD/mutTK(SR39)repADP在晚期胰腺癌的临床试验中也显示出有效性 [37]

  • 3.2.2 腺病毒载体递送免疫调节基因

  • 腺病毒载体还可以通过递送表达免疫调节基因来刺激患者的抗肿瘤免疫反应。研究已证实搭载干扰素IFN-β 或IFN-α-2b基因的腺病毒载体治疗恶性胸膜间皮瘤的安全性。使用腺病毒IFN-α-2b载体联合塞来昔布和吉西他滨的抗肿瘤治疗正在进行Ⅲ期试验 [38, 39]。粒细胞-巨噬细胞集落刺激因子(Granulocyte-macrophage Colony-stimulating Factor, GM-CSF)被认为是诱导免疫细胞活化以触发抗肿瘤反应的关键因子。单用搭载GM-CSF基因的选择性复制腺病毒嵌合载体(Oncos-102,HAd5/3-Δ24-GM-CSF)或联用PD-L1单抗(Durvalumab) 和自体树突状细胞免疫疗法(DCVAC/PCa)正用于实体肿瘤治疗的临床研究 [40]。CG0070也是一种可选择复制的溶瘤腺病毒载体,使用人E2F-1启动子控制表达GM-CSF以靶向杀灭膀胱癌细胞。该载体的Ⅱ期临床试验中期结果表明其毒性水平可接受,注射病毒6个月后,所有患者的完全缓解率为47%[41]。CD40是一种细胞表面受体,一旦与CD40配体(CD40L)相互作用,就可以阻止细胞增殖和促进细胞凋亡。由于CD40在大多数乳腺癌细胞中过度表达,研究者使用腺病毒载体搭载乳腺癌细胞特异性启动子所控制的CD40L基因(AdEHCD40L) 靶向杀灭乳腺癌细胞 [42]。LOAd703是利用HAd5/35嵌合腺病毒载体共表达三聚体CD40L和免疫增强多肽4-1BBL来治疗胰腺癌 [43]

  • 3.3 嵌合衣壳和组织亲和性改造的腺病毒溶瘤载体

  • 腺病毒主要的细胞受体CAR通常在肿瘤细胞上呈低水平表达,因此研究者们试图寻找替代的腺病毒-细胞结合位点。DNX-2401是一种选择性复制的腺病毒嵌合载体,在该载体的纤突蛋白中插入了一段ACDCRGDCFCG肽序列(RGD4C),可以与整合素(Integrin)αvβ3和 αvβ5结合,以介导腺病毒不通过与CAR受体结合而侵入细胞内部 [44]。该载体已在复发性恶性胶质瘤患者中进行了临床测试。具有相似结构特点的腺病毒载体还包括ICOVIR-5和ICOVIR-7 [45]。VCN-01腺病毒载体的纤突蛋白内RGDK氨基酸序列被硫酸肝素糖胺聚糖(Glycosaminoglycan, HSG) 结合位点(KKTK)所取代,以通过细胞表面糖胺聚糖受体介导病毒入胞。该载体还搭载人糖基磷脂酰肌醇锚定酶(Glycosylphosphatidylinositolanchored Enzyme) 和PH20透明质酸酶(PH20hyaluronidase),以促进病毒在实体瘤间质中的传播。VCN-01已被证明对胰腺癌等癌症有效 [46]。 HAd5/3嵌合载体是将HAd5溶瘤腺病毒载体的纤突顶球(Fiber Knob) 结构域置换为HAd3腺病毒的相应结构域。后者的顶球结构域更适合与肿瘤细胞表面高表达的Ad3受体结合,使HAd5/3嵌合载体通过Ad3受体进入肿瘤细胞内部 [47]。前文提到的Oncos-102载体就是一种针对实体瘤的HAd5/3嵌合病毒。另一种肿瘤选择性腺病毒嵌合载体ColoAd1(Enadenotucirev)是通过对不同血清型腺病毒的重组筛选演化而来,该病毒载体表达B亚群的Ad11p衣壳骨架,基因组中E3和E4缺失, E2B区域为Ad3/Ad11p嵌合序列 [48]。ColoAd1在多个实体瘤中显示出具有强大而特异的肿瘤细胞杀伤特性 [49]

  • 4 腺病毒载体在基因治疗应用中面临的机遇与挑战

  • 腺病毒作为基因治疗载体具有很多优点:(1) 在各种细胞中具有较高的基因转导效率;(2)病毒基因组DNA不整合入宿主细胞染色体中;(3)对不同靶组织具有广泛的亲和性;(4)可以被大规模工业化生产等 [1]。目前种类繁多的腺病毒载体正在被用于各类临床试验中,显示出令人鼓舞的安全性和有效性。但是,腺病毒基因治疗载体的主要挑战是克服在人群中广泛存在的抗腺病毒免疫机制,对病毒衣壳蛋白强大的固有免疫反应,以及新合成的病毒和转基因产物引起的获得性免疫反应。由于自然条件下腺病毒的广泛流行,受试者体内存在的中和抗体可显著削弱HAd5载体的基因转导能力,严重影响包括HAd5在内的人腺病毒载体的应用 [50]。因此,正确控制宿主免疫应答是确保该疗法成功的关键。近年来,研究者尝试开发基于“稀有”血清型的人腺病毒作为基因治疗用载体等来规避人群中存在的HAd5中和抗体 [51]。同时开发非人类腺病毒载体(如黑猩猩腺病毒ChAd3)也成为主要的研究方向 [52]。此外,高容量HCAd载体因可最大限度地限制病毒蛋白的残留表达从而抑制宿主的免疫反应,使其可以在体内实现长期的转基因表达,也将是该领域的发展方向 [53]。HCAd载体还因巨大的装载容量,可以搭载目前被广泛应用的基因编辑系统,如CRISPR/Cas9, ZFNs和TALENs等,以实现对致病基因的精准编辑和改造。

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