Gene therapy of inherited diseases
Gene therapy of inherited diseases
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DOI：
10.1016/s0140-6736(08)60874-0
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发表时间：
2008-06-14
期刊：
影响因子：
168.9
通讯作者：
Cavazzana-Calvo, Marina
中科院分区：
文献类型：
--
作者：
Fischer, Alain;Cavazzana-Calvo, Marina
成千上万的罕见疾病是由孟德尔基因错误引起的。到目前为止，已经确定了1800多个与罕见疾病相关的基因，在许多情况下，它们的表达模式和功能已经被揭开。这些信息是制定治疗策略的先决条件。根据疾病的严重程度，并通过评估可行性和治疗方案，在某些情况下可以将基因治疗视为一种选择（表2）。遗传性疾病在表型上具有很大的异质性。在考虑合适的基因治疗形式时，需要确定三个基本参数：突变是否导致功能的丧失或获得；基因产物的功能是否影响细胞的存活或发育；以及疾病基因的组织特异性。有四种不同的基因治疗策略。首先，加入突变基因的正常拷贝。这种方法最适合于功能丧失突变，也是迄今为止大多数基因治疗尝试的焦点。第二，对信使RNA进行修饰以避免突变的后果。当突变的外显子不是必不可少的时候，这种策略可以被视为一种有希望的选择。第三，抑制突变基因的表达。这种方法可能有助于阻止功能获得蛋白的表达或抑制隐剪接位点，从而防止具有有害后果的异常剪接产物的表达。如果意想不到的毒性作用（如在肝脏中所见）不严重，使用小干扰RNA似乎是首选。最后，基因修复——一种旨在恢复突变的终极而优雅的策略。该技术基于使用由DNA序列特异性结合域和能够诱导DNA中位点特异性双链断裂的内切酶组成的嵌合蛋白。同时，一个包含与突变DNA片段相对应的野生型序列的模板被引入细胞，并作为同源重组修复的底物。与FOK1核酸酶偶联的锌指蛋白结构域已被工程化，并在5-17%的细胞中特异性纠正编码细胞因子受体γ -c链的IL2RG基因（如x连锁严重联合免疫缺陷[SCID-X1]）。然而，由于许多技术问题，临床应用之路仍然很长。RNA病毒（如γ逆转录病毒、唾液病毒和慢病毒）正被用于介导整合性基因转移。8-10既可以使用病毒启动子，如逆转录病毒长末端重复序列，也可以使用强效病毒或细胞内部启动子。γ逆转录病毒载体仅在分裂细胞中有效，因为整合前复合物不能穿过核膜，而慢病毒载体可以将基因传递到非分裂细胞的基因组中。对原病毒衍生载体的整合位点已经给予了大量的关注。逆转录病毒载体在CpG岛附近整合频率很高，11-13意味着长末端重复序列也可以靶向活性基因，尽管没有选择性向转录起始位点的趋向性。源自HIV或猴免疫缺陷病毒的慢病毒样载体可以靶向活性基因。14,15完整的逆转录病毒
Thousands of rare diseases are caused by a Mendelian genetic error. So far, more than 1800 genes associated with rare diseases have been identified1 and, in many cases, their expression patterns and functions have been unravelled. This information is a prerequisite for development of a therapeutic strategy. Depending on the disease’s severity, and by assessing feasibility and treatment alternatives, gene therapy can be viewed as an option in some instances (table2). Genetic disorders are phenotypically very heterogeneous. In considering the appropriate form of gene therapy, three basic parameters need to be established: whether a mutation leads to a loss or gain of function; whether or not a gene product’s function affects cell survival or development; and the disease gene’s tissue specificity. 3 There are four different gene therapy strategies. First, addition of a normal copy of the mutated gene. This approach is best suited to loss-of-function mutations and has been the focus of most gene therapy attempts so far. Second, modification of messenger RNA to avoid the consequences of mutation. This strategy can be viewed as a promising option when the mutated exon is not indispensable. 4 Third, inhibition of expression of a mutated gene. This approach is potentially useful to prevent the expression of a gain-of-function protein or to inhibit a cryptic splice site, thus preventing expression of an abnormally spliced product with deleterious consequences. The use of small interfering RNA seems to be preferred, 5 provided that unexpected toxic effects (as seen in the liver6) are not serious. Last, repair of the gene—an ultimate and elegant strategy aimed at reverting mutation. 7 This technology is based on the use of chimeric proteins composed of a DNA-sequence-specific binding domain and an endonuclease capable of inducing site-specific double-strand breaks in DNA. Simul taneously, a template encompassing the wild-type sequence that corresponds to the mutated stretch of DNA is introduced into the cell and acts as a substrate for repair by homologous recombination. Zinc-finger protein domains coupled to the FOK1 nuclease have been engineered and shown specifically to correct the IL2RG gene encoding the γc-chain of cytokine receptors (as noted in X-linked severe combined immunodeficiency [SCID-X1]) in 5–17% of cells. 7 However, the road to clinical application is still long, because of many technical concerns.RNA viruses (eg, γ retroviruses, spumaviruses, and lentiviruses) are being used to mediate integrative gene transfer. 8–10 Either a viral promoter, such as the retroviral long-terminal repeat, or a potent viral or cellular internal promoter can be used. γ retroviral vectors are only effective in dividing cells, because the preintegration complex cannot cross the nuclear membrane, whereas lentiviral vectors can deliver genes to the genome of non-dividing cells. Substantial attention has been paid to the integration sites for provirus-derived vectors. Retroviral vectors integrate with a high frequency near CpG islands, 11–13 meaning that the long-terminal repeat can also target active genes albeit without a selective tropism to the transcription start site. Lentivirus-like vectors derived from HIV or simian immunodeficiency virus can target active genes. 14, 15 Intact retroviral