Replication-defective herpes simplex virus vectors for gene transfer in vivo
Replication-defective herpes simplex virus vectors for gene transfer in vivo
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DOI：
10.1073/pnas.93.21.11319
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Glorioso, JC
中科院分区：
文献类型：
--
作者：
Marconi, P;Krisky, D;Glorioso, JC
单纯疱疹病毒1型具有许多生物学特性，这表明它可以被设计成直接将治疗性基因转移到神经元的载体。这些特征包括：(I)它自然具有建立长潜伏期的能力，在这种状态下，病毒基因组没有整合，裂解基因处于静止状态，宿主细胞的代谢功能明显不受干扰；(Ii)潜伏期相关转录本(LAT)的表达由神经元特异的、潜伏期活跃的启动子(LAP)元件驱动，这可能被证明有助于表达潜伏的病毒基因组中的转基因；以及(Iii)观察到，通过删除必要基因而产生的复制缺陷突变体保持了在神经系统中建立潜伏状态的能力(1)。此外，81个单纯疱疹病毒(HSV)基因中的许多基因在细胞培养中不是病毒复制所必需的，可以方便地删除，以便为大量外源DNA的掺入提供空间，而且几乎所有的病毒基因都是连续的单位，使得遗传操作变得可行。病毒也可以培养到高滴度，病毒的感染力非常有效。影响HSV有效载体发展的主要因素是缺陷载体的残留细胞毒性和有限的转基因表达时间。即使是复制能力不强的突变病毒也是细胞毒性的，在体外很容易杀死神经元，除了HSV LAP元件外，病毒和外来启动子似乎受到病毒快速诱导启动子关闭机制的控制。已经开发了两种不同类型的基于HSV的基因递送系统。第一种类型由基因工程基因组载体组成，这些载体可能会在病毒在有丝分裂后细胞(如神经元)中复制所需的基因中缺失，或者可能是完全复制缺陷的，需要互补才能进行载体传播。第二种基于单纯疱疹病毒的载体系统，称为扩增子，使用有缺陷的辅助病毒突变体包装含有单纯疱疹病毒来源的DNA合成和包装序列的连体质粒。我们一直致力于复制缺陷基因组载体的开发。在编码ICP4(如D120)(2)的单个必需即刻早期(IE)基因中，缺失了“第一代”缺陷基因组载体。这些载体可以在ICP4互补的细胞系中繁殖，但感染神经元后，病毒基因的表达在IE基因表达水平上被中止。虽然这些载体的致病性较低，可用于在大脑中有效地转移和瞬时表达报告基因(见下文)，但它们对培养中的神经元是有毒的，会产生细胞质起泡、宿主细胞DNA碎裂和染色体异常等细胞病变效应(3)。推测残留的细胞毒性是由于HSV基因产物的表达，因为紫外线照射的病毒颗粒是无毒的，而干扰IE基因表达的干扰素治疗显著降低了细胞毒性。虽然ICP4编码基因的缺失会导致早期和晚期病毒基因的表达中断，但在没有ICP4的情况下，其他四种直接早期基因产物和ICP6(核糖核苷酸还原酶大亚基)都会过度表达。ICP4、ICPO、ICP27和ICP22在稳定的转染试验中都被证明是有毒的(4)，因此可能需要联合删除这些基因以消除毒性。UL41虽然不是IE基因的产物，但它存在于病毒粒子中，并通过破坏宿主细胞基因的不稳定而导致宿主细胞蛋白质合成的中断[5]；此外，UL41还可以减少HSV和…的转基因表达
Herpes simplex virus 1 has a number of biological features which suggest that it could be engineered as a vector for direct transfer of therapeutic genes to neurons. These features in-clude (i) its natural ability to establish life-long latency, a state in which the viral genome is not integrated, lytic genes are quiescent, and the metabolic functioning of the host cell is apparently undisturbed;(ii) the expression of latency-associated transcripts (LATs) driven by neuron-specific, laten-cy-active promoter (LAP) elements, which may prove useful in expressing transgenes from latent viral genomes; and (iii) the observation that replication-defective mutants created by the deletion of essential genes retain the ability to establish a latent state in the nervous system (1). In addition, many of the 81 herpes simplex virus (HSV) genes are not required for viral replication in cell culture and may conveniently be deleted to provide space for incorporation of substantial foreign DNA, and almost all viral genes are contiguous units, making genetic manipulation feasible. The virus can also be grown to high titer, and viral infectivity is very efficient. The major imped-iments to the development of HSV-effective vectors relate to residual cytotoxicity of defective vectors and the limited duration of transgene expression. Even replication-incompetent mutant viruses are cytotoxic, readily killing neurons in vitro, and with the exception of the HSV LAP elements, viral and foreign promoters appear to come under control of the virus' ability to rapidly induce mechanisms of promoter shutoff. Two different types of HSV-based gene delivery systems have been developed. The first type consists of genetically engineered genomic vectors, which may be deleted in genes required for the virus to replicate in postmitotic cells such as neurons or may be completely replication-defective, requiring complementation for vector propagation. The second type of HSV-based vector system, referred to as amplicons, uses defective helper-virus mutants for packaging concatemeric plasmids containing an HSV origin of DNA synthesis anda packaging sequence. We have focused our efforts on the development of replication-defective genomic vectors. The" first generation" defective genomic vectors were deleted in the single essential immediate early (IE) gene encod-ing ICP4 (eg, d120)(2). These vectors can be propagated in ICP4-complementing cell lines, buton infection of neurons, viral gene expression is aborted at the level of IE gene expression. Although these vectors are of reduced pathogenicity and can be used to efficiently transfer and transiently express reporter genes in brain (see below), they are toxic to neurons in culture, producing cytopathic effects such as cyto-plasmic blebbing, host cellDNA fragmentation, and chromo-somal aberrations (3). It is presumed that residual cytotoxicity results from the expression of HSV gene products, because UV-irradiated viral particles are not toxic and interferon treatment to disrupt IE gene expression markedly reduces cytotoxicity. Although deletion of the gene coding for ICP4 aborts the expression of both early and late viral genes, the other four immediate early gene products and ICP6, the ribonucleotide reductase large subunit, are overexpressed in the absence of ICP4. ICP4, ICPO, ICP27, and ICP22 have all been shown to be toxic in stable transfection assays (4), so deletion of these genes in combination may be required to eliminate toxicity. UL41, although notan IE gene product, is present in the virion and is responsible for shutoff of host cell protein synthesis through destabilization of host cell mRNA (5); in addition, UL41 many reduce transgene expression from HSV …