Development of non-integrating lentiviral vectors for safer gene therapy

在过去的几年中，基因疗法已被用于治疗儿童腺苷脱氨酶（ADA）缺乏症和常见？-链，或x连锁，严重联合免疫缺陷（SCID）。逆转录病毒载体被用来传递治疗基因，该基因能够整合到宿主细胞的基因组中并治愈疾病。不幸的是，在法国的一项x连锁SCID试验中，三名儿童在将逆转录病毒DNA整合到人类基因组后患上了T细胞白血病。这些患者的白血病是由治疗基因整合到宿主细胞染色体的危险位置引起的。基因整合到这个位置引起了一个叫做插入突变的事件，导致细胞转化和癌症。这项临床试验的结果强调了开发更安全的基因治疗载体的必要性。这可以通过防止治疗基因整合到患者基因组中不想要的和危险的区域来实现。我们小组研究的不是逆转录病毒，而是慢病毒作为基因治疗载体。慢病毒可以包装大量的DNA，与逆转录病毒不同，它们既可以感染非分裂细胞，也可以感染分裂细胞。这两个特性意味着经过适当改造的慢病毒在治疗许多组织和器官类型的遗传疾病方面具有巨大的潜力。像逆转录病毒一样，慢病毒会整合到患者自身的基因组中。这是有益的，因为这意味着理论上只需一次使用慢病毒就可以实现对遗传缺陷的长期纠正。然而，如上所述，存在破坏患者基因组中正常DNA的风险。因此，我们建议设计慢病毒，以消除这种插入突变的风险，同时保留所有使慢病毒成为基因治疗有前途的载体的有益特性。插入突变可以通过抑制慢病毒DNA整合到患者基因组中来预防。慢病毒通过整合酶蛋白进行整合。我们正在用一种改变了的、无效的整合酶蛋白制造慢病毒。这些新设计的慢病毒将被测试其安全将基因传递到各种非分裂细胞类型的能力。由于细胞不分裂，我们预计新传递的基因将留在细胞中，但不会整合到细胞的基因组中。这种治疗性基因将作为一个独立的DNA片段保存，不会干扰正常的细胞DNA功能。我们预计这些非整合慢病毒载体在视网膜和肌肉等细胞中最有用。此外，由于治疗基因将在细胞内无限期地维持，因此只需要一次剂量的慢病毒。这样的系统将转化为一种比目前可用的基因治疗技术更安全、更简单的方法。

During the last few years gene therapy has been used to treat children with adenosine deaminase (ADA) deficiency and common ?-chain, or X-linked, severe combined immunodeficiency (SCID). Retroviral vectors were used to deliver the therapeutic gene, which was able to integrate into the genome of host cells and cure the disease. Unfortunately, three children in a French trial for X-linked SCID have developed T cell leukaemia following the integration of retroviral DNA into the human genome. The leukemia in these patients was caused by the therapeutic gene integrating into a dangerous location in a host cell chromosome. Integration of the gene into this location caused an event called insertional mutagenesis and resulted in cell transformation and cancer. The outcome of this clinical trial emphasizes the need to develop safer gene therapy vectors. This can be achieved by preventing the integration of a therapeutic gene into an unwanted and dangerous area of the patients' genome. Instead of retroviruses, our group studies lentiviruses as gene therapy vectors. Lentiviruses can package large amounts of DNA, and unlike retroviruses they can infect non-dividing as well as dividing cells. These two properties mean that properly engineered lentiviruses will have vast potential to treat genetic diseases of many tissue and organ types. Like retroviruses, lentiviruses integrate into the patients own genome. This is beneficial because it means that long-term correction of a genetic defect can theoretically be achieved with just one administration of the lentivirus. However, as discussed above, there is a risk of disrupting normal DNA in the patient's genome. Therefore, we have proposed to engineer lentiviruses that eliminate this risk of insertional mutagenesis while retaining all the helpful properties that make lentiviruses such a promising vehicle for gene therapy. Insertional mutagenesis can be prevented by inhibiting integration of the lentivirus DNA into the patient's genome. Lentiviruses integrate by using an integrase protein. We are in the process of generating lentiviruses with an altered, ineffective integrase protein. These newly engineered lentiviruses will be tested for their ability to safely deliver genes into various non-dividing cell types. Since the cells do not divide, we expect the newly delivered gene to stay in the cell but not to integrate into the cell's genome. This therapeutic gene will be maintained as a separate piece of DNA that will not interfere with normal cellular DNA functions. We expect these non-integrating lentivirus vectors to be most useful in cells such as retina and muscle. Furthermore, since the therapeutic gene will be maintained indefinitely inside the cell, only one dose of the lentivirus is necessary. Such a system will translate into a safer and simpler approach than the currently available gene therapy techniques.