腺相关载体毒性，生存还是毁灭？

StephenW

Editorial

Molecular Therapy (2015); 23 11, 1673–1675. doi:10.1038/mt.2015.182
Adeno-associated Vector Toxicity—To Be or Not to Be?

Hildegard Büning and Manfred Schmidt

    Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
    National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ) Department of Translational Oncology, Heidelberg, Germany

Vectors based on adeno-associated virus (AAV) are currently in use in numerous gene therapy clinical trials with reports of benefit for patients, for example in treating hemophilia or inherited blindness. An AAV1 vector (Glybera) has recently been approved as the first gene therapy to achieve marketing authorization in Europe. More than 120 clinical trials using AAV have shown vector safety, with a transient and asymptomatic hepatitis as the most severe side effect. In comparison to the widespread use of AAV vectors in clinical, preclinical, and basic research, there has been limited study of the interaction of the parental virus in humans. Aside from reports suggesting an association between AAV infection early in pregnancy and spontaneous abortions,1 AAV infection has so far not been associated with illness. However, in a recent issue of Nature Genetics, Nault et al. report the presence of AAV insertions in a total of five different cancer driver genes in 11 of 193 patient samples of hepatocellular carcinoma (HCC) tumors compared with their matched nontumor liver tissue.2 Although the presence of AAV sequences in tumor samples may not be very surprising given the high prevalence of AAV infection in humans, the validity of the conclusion for an active role of AAV in tumor formation—in particular, the extrapolation of these results to AAV vectors—, has to be challenged.

AAV is a nonenveloped, single-stranded DNA virus, with a biphasic life cycle, with productive replication in the presence of a helper virus and establishment of a latent infection in its absence.3 Cell culture experiments pointed toward viral genome integration with preference for a specific region on human chromosome 19 (AAVS1), now known as a safe harbor for genetic engineering.3 Of the three initially discovered serotypes (AAV1, AAV2, AAV3), AAV2 was the first to be vectorized. Current systems of AAV vector production use vector genome and packaging signals (ITRs, inverted terminal repeats) built on AAV2, whereas the capsid is chosen depending on the desired cell type or tissue preference and/or antigenic reactivity.3 At least 13 human and nonhuman primate AAV serotypes have been identified so far, and rational-design and directed evolution–based strategies have further expanded the “AAV toolbox,” which comprises a growing number of naturally occurring and engineered capsids into which vector genomes either of the single-stranded (4.5 kilobases, natural) or self-complementary (2.3 kilobases, artificial) design are encapsidated.3,4

The prevalence of AAV infection in the human population is high, reaching >70% for serotype 2.5 Latent infection is established, with liver, bone marrow, spleen, and gut appearing as predominant tissues.6 To date, no human pathology has been associated with AAV infection. On the contrary, a protective role of AAV against human papillomavirus–induced carcinomas,7 as well as induction of antiproliferative gene clusters upon AAV infection, was reported.8 Nault et al. sought to identify factors contributing to the development of HCC in nonfibrotic livers, a rare subset of this disease with frequencies of around 5% of all HCCs.2 Upon screening telomerase reverse transcriptase (TERT) promoter sequences, commonly altered in HCC, they detected a 208–base pair (bp) sequence of the wild-type AAV2 3′-ITR upstream of the start codon of TERT. The insertion was accompanied by a 16-bp deletion of the human sequence and a further 7-bp insertion of unknown origin. TERT mRNA was elevated in this HCC sample compared with nonmalignant tissue of the same liver, consistent with a role for telomerase in malignant transformation. Prompted by this finding, researchers screened a total of 193 HCCs and their matched nontumor liver tissue—but no liver biopsy samples from healthy subjects—for the presence of AAV. The majority of these tissues (147/193) lacked AAV sequences. However, in 11 of the 193 HCC tumors, AAV insertions were found in five different cancer driver genes (TERT (1/11), cyclin A2 (CCNA2) (4/11), cyclin E (CCNE1) (3/11), tumor necrosis factor superfamily member 10 (TNFSF10) (2/11) and lysine-specific methyltransferase 2B (KMT2B) (1/11)).

The authors’ interpretation of these data requires scrutiny. In total, 11 HCCs were found to harbor deleted AAV sequences, although only 6 of these 11 were found in patients lacking other known risk factors such as hepatitis virus B or C infection or alcohol consumption (Supplementary Table 2 in the article by Nault et al.). Notably, 4 of these 6 HCCs presented AAV-independent HCC-related mutations. Thus, only 2 HCCs were identified with AAV as the single detected “mutation.” It is not shown in detail whether these 2 HCCs also showed significant HCC gene–related RNA overexpression (Figures 1b and 2b in the article by Nault et al.). Thus, it remains to be determined whether AAV is simply a passenger mutation rather than a driver mutation in HCC formation.

The extrapolation of the findings to suggest AAV-related genotoxicity must also be challenged. A complete virus genome was detected in none of the samples. Indeed, the fragments with homology to AAV were all rather small. Furthermore, no distinct integration pattern emerged, with insertions appearing upstream of a promoter sequence (1/11), within an intron (5/11), within an exon (2/11), or within the 5′- or 3′–untranslated region (3/11). Thus, in the absence of a complete AAV genome and a more unifying pattern, it appears a bit exaggerated to attribute any genotoxicity to AAV rather than to other foreign DNA (if any) integrated into these regions.

Two recent publications have confirmed preference for AAV2 to integrate into the AAVS1 region on chromosome 19, which is caused by the accumulation of Rep binding sites and terminal resolution site (trs)-like motifs within this region.9,10 It is thus surprising that no AAV sequences were found within the AAVS1 region in either the HCCs or the matched nonmalignant tissue,2 even though wild-type AAV has an active Rep integrase targeting this site.

Finally, the implications of these results for the use of AAV vectors in the clinic appear quite limited. Current AAV vectors are “gutless,” i.e., devoid of all known viral open reading frames, leaving the ITRs as the sole viral sequences in cis. The wild-type AAV2 3′-ITR is may be the only common denominator in the reported HCC integrations, as it was detected in 10 of the 11 cases.2 This sequence, however, is not present in the commonly used AAV vector genomes, as during the cloning procedure for AAV’s vectorization parts of the wild-type 3′-ITR were replaced by the 5′-ITR.11

Furthermore, AAV vector genomes tend to persist episomally as concatemeric structures.3 Although numerous groups have demonstrated the safety of AAV vectors despite rare integration events,12,13,14,15,16,17 a handful of preclinical studies have suggested a genotoxic potential for AAV vector integration within hepatic tissue under specific circumstances.18,19,20 A thorough analysis of experimental variables identified neonatal injection of a high dose of hepatotropic AAV8 vectors in combination with a strong enhancer/promoter sequence as critical parameters,19 arguing against an intrinsic ITR-related genotoxicity. The sensitivity of neonatal mice to insertion at Rian, identified as an integration hot spot, was correlated with accessibility of Rian at this stage of development. Insertion at Rian per se—even in neonatal mice—did not appear to be genotoxic on its own, but rather required use of a strong promoter/enhancer.19 Of further note, integrations that were found to be associated with HCC in mice clustered at the Mir341 locus in Rian, which lacks a human orthologue.19

None of the other existing preclinical and clinical AAV studies has demonstrated any severe adverse events.13,17,21,22,23 Associated integration site studies also did not find any relevant integration preferences in HCC-susceptible gene regions. Accordingly, recently presented integration data from different biopsy specimens from nonhuman primate tissues (liver, spleen, adrenal gland) after intravenous administration of clinical AAV5-cohPBGD vectors (unpublished clinical data) revealed no unwanted side effects or integration at these loci.24 To conclude, we do not aim to challenge the authors’ findings of partial wild-type AAV2 sequences in 11 out of 193 cases of human HCCs. However, we believe that the implications of the findings are significantly overestimated, at least with regard to AAV vectors, whose genome sequences are profoundly distinct from that of wild-type AAV2. The vast majority of preclinical and all clinical studies have shown the genome-wide distribution of rare AAV vector integrations without any genotoxicity or preference for the previously reported HCC-associated genes, emphasizing the very limited genotoxic risk of AAV vectors.

StephenW

社论

分子疗法（2015年）; 23 11，1673年至1675年。 DOI：10.1038 / mt.2015.182
腺相关载体毒性，生存还是毁灭？

希尔德加德Büning和曼弗雷德·施密特

    实验血液学，汉诺威医学院，汉诺威，德国研究所
    国家中心肿瘤疾病（NCT）和德国癌症研究中心（DKFZ）转化肿瘤科，海德堡，德国

基于腺相关病毒（AAV）载体，目前在许多基因治疗临床试验使用具有益处的患者报告，例如，在治疗血友病或继承失明。源自于AAV2载体（Glybera）最近被批准作为第一基因治疗来实现在欧洲上市许可。采用AAV 120多名临床试验表明载体的安全性，有一过性和无症状性肝炎的最严重的副作用。相比于临床，临床前，和基础研究的广泛使用AAV载体的，出现了在人类中的亲代病毒的相互作用的有限的研究。除了报告显示AAV病毒感染在怀孕早期自然流产，1 AAV感染之间的关联至今尚未与疾病有关。然而，在最近的自然遗传学，Nault等人的问题。报告中一共有五个不同癌症驱动基因在11 193的患者的肝细胞癌样本AAV插入的存在下与它们的匹配的非肿瘤肝tissue.2相比（HCC）的肿瘤虽然AAV序列的肿瘤样品中的存在可能不是很奇怪，因为AAV感染人类的​​高患病率，得出的结论为AAV在肿瘤的积极作用的有效性地层中特别是，这些结果对AAV vectors-外推，有受到挑战。

AAV是一个无包膜，单链DNA病毒，具有双相生命周期，与在辅助病毒和建立在其absence.3细胞培养实验潜伏感染的存在下生产的复制具有优先要指出朝向病毒基因组的整合人类19号染色体（AAVS1）特定的区域，现在被称为一个避风港遗传engineering.3三个最初发现血清型（AAV1，AAV2，AAV3）中，AAV2是第一个被量化。建立在AAV2 AAV载体生产使用载体基因组和包装信号序列（ITR，末端反向重复序列）的现有系统，而根据所需的细胞类型或组织偏爱和/或抗原reactivity.3至少13人和非人衣壳被选择灵长类动物AAV血清已被确定为止，合理设计和定向进化的基于策略已进一步扩大了“AAV工具箱”，它包括有越来越多的天然存在的和工程改造衣壳到其中载体基因组的任一单链（ 4.5千碱基，自然），或自补（2.3万个碱基，人为的）设计都encapsidated.3,4

AAV感染的人群中的患病率是高，达到> 70％的血清型2.5潜伏感染成立，肝脏，骨髓，脾和肠出现作为主要tissues.6迄今为止，还没有人体病理学一直伴随AAV感染。与此相反，AAV的时AAV感染的保护作用的抗增殖的基因簇的抗人类乳头瘤病毒诱导的癌，7以及诱导，是reported.8 Nault等。寻求确定因素导致肝癌的nonfibrotic肝脏的发展，本病以大约5％的HCCs.2经筛选端粒酶逆转录酶（TERT）启动子序列，通常改变在肝癌的频率罕见的子集，他们检测208个碱基对（bp）的序列的野生型TERT的起始密码子的AAV2 3'- ITR上游。插入伴随着一个16 bp的缺失人类序列和来历不明的又一个7个碱基插入。 TERT表达升高这一肝癌样本中使用相同的肝脏良性组织，对端粒酶在恶性转化的作用是一致的比较。由这一发现提示，研究员从健康受试者换的AAV存在共193肝癌和它们匹配的非肿瘤肝组织，但是没有肝活检样品进行筛选。大多数这些组织（一百九十三分之一百四十七）的缺乏AAV序列。但是，在193肝癌肿瘤11，AAV插入被发现在五个不同的癌症驱动基因（TERT（1/11），细胞周期蛋白A2（CCNA2）（4/11），细胞周期蛋白E（CCNE1）（3/11），肿瘤坏死因子超家族成员10（TNFSF10）（2/11）和赖氨酸特异性甲基2B（KMT2B）（1/11））。

这些数据的作者的解释需要推敲。总体而言，11肝癌被发现怀有删除AAV序列，虽然只有6这11个被发现在缺少其他已知的危险因素，如乙型肝炎病毒或肝炎感染或饮酒（补充表2中的文章Nault等患者）。值得注意的是，其中4 6肝癌呈现AAV-独立的肝癌相关基因突变。因此，只有2肝癌鉴定与AAV作为单一检测“突变”。它没有详细示出这些2肝癌是否也表现出显著肝癌基因相关的RNA过表达（图1b和2b中的文章中由Nault等人）中。因此，仍有待确定的AAV是否是一个简单的从属突变，而不是一个驱动突变在HCC形成。

调查结果的推断表明AAV相关的基因毒性也必须受到挑战。在没有任何样品的检测到完整的病毒基因组中。事实上，与同源性AAV的片段都比较小。此外，没有任何明显的集成的模式出现，与插入出现的启动子序列（1/11）的上游，一个内含子（5/11）中，外显子（2/11）之内，或在5'或3'-翻译区（3/11）。因此，在没有一个完整的AAV基因组和一个更加统一图案，看来有点夸张归因任何遗传毒性AAV而非其它的外源DNA（如有的话）集成到这些区域。

两个近期的出版物证实偏爱AAV2融入AAVS1区19号染色体上，它是由众议员结合位点和终端解决网站（TRS）般的图案堆积在此region.9,10它是这样令人惊讶的是没有AAV序列的AAVS1区域内无论是在肝癌或匹配的非恶性组织中发现，2尽管野生型腺相关病毒有一个活跃的声誉整合瞄准了这个网站。

最后，这些结果对于在临床使用AAV载体的影响似乎相当有限。目前AAV载体是“无胆”，即缺乏所有已知病毒的开放阅读框，使国际电信规则中顺的唯一病毒序列。野生型AAV2 -3'- ITR是可能在所报告的肝癌集成唯一的共同点，因为它是在10 11 cases.2该序列的检测到的，但是，不存在于常用的AAV载体基因组，作为野生型AAV的矢量份克隆过程期间3'- ITR所取​​代5'- ITR.11

此外，AAV载体基因组倾向于坚持附加体作为concatemeric structures.3尽管许多研究小组已经证明，尽管罕见一体化事件的AAV载体的安全性，12,13,14,15,16,17少数临床前研究表明遗传毒性潜力根据具体circumstances.18,19,20肝组织内AAV载体的整合实验变量的深入分析发现新生儿注射高剂量的嗜肝AAV8载体，结合强大的增强子/启动子序列的关键参数，19驳斥了内在ITR相关的基因毒性。新生小鼠对插入的感光度日安，确定为一个集成的热点，率与日安的访问，在这个发展阶段。在插入日安本身，甚至在新生小鼠，没有出现具有遗传毒性自己，而是需要使用强启动子/ enhancer.19还值得注意，积分被发现能与肝癌的聚集在小鼠相关的所述Mir341轨迹中日安，它缺乏人类orthologue.19

没有任何其他现有的临床前和临床AAV研究已经证明相关的整合位点的研究还没有发现肝癌易感基因区域任何有关整合的偏好任何严重不良events.13,17,21,22,23。从临床AAV5-cohPBGD载体（未发表的临床数据）静脉内给药后非人灵长类组织（肝，脾，肾上腺）不同活检标本因此，最近提出的集成数据显示没有有害的副作用或整合这些loci.24总括我们的目的不是在11出193例人肝癌的挑战部分野生型AAV2序列的作者的调查结果。但是，我们认为，该研究结果的影响是显著高估，至少对于AAV载体，其基因组序列是深刻的从野生型AAV2截然不同。临床前和所有临床研究绝大多数都表现出罕见的AAV载体整合全基因组分布，而不对此前公布的肝癌相关基因的任何遗传毒性或偏好，强调AAV载体非常有限的遗传毒性风险。