Precision genome editing with tandem autologous transplantation as a therapy for multiple severe immune-mediated diseases

自体造血干细胞移植(ASCT)是治疗严重免疫沉降性疾病(IMD)的重要手段。最近的研究表明，它对治疗多发性硬化症(MS)和硬皮病等严重的IMD是安全有效的。这包括从患者身上收集骨髓来源的干细胞，然后使用化疗和治疗性抗体的组合引发严重但短暂的免疫抑制。这会根除很大一部分免疫系统，包括自身反应细胞。然后，骨髓来源的干细胞被重新注入，免疫系统重新构建，长期副作用最小。欧洲骨髓移植登记数据显示，超过2500名患者接受了IMD的自体移植。自2005年以来，ASCT女士自体移植后只有一例死亡，在诱导长期缓解方面优于MS的所有其他治疗方法，也是唯一被发现可减少残疾的治疗方法。在硬皮病中，ASCT导致了非常显著的生存改善(6年时86%对51%)。虽然ASCT可以阻止许多患者的疾病进展，但30%-50%的患者最终会重新发展为原来的IMD，5%-10%的患者会发展为不同的IMD。因此，这一治疗策略有很大的改进余地。近年来，由于CRISPR-Cas9等可编程核酸酶的出现，分子生物学发生了一场革命，因为它们允许我们对基因组序列进行精确的改变。它们很可能在未来十年内成为极其重要的治疗工具。我们正在使用这项技术来修改骨髓来源干细胞的基因组序列，以治疗遗传性血细胞生产和免疫系统疾病。下一步是使用这种方法来治疗获得性免疫系统疾病，如IMD。大规模的遗传分析揭示了一种目标基因，该基因对包括多发性硬化症(MS)、硬皮病、类风湿性关节炎和克罗恩病在内的20种不同的IMD具有广泛的保护作用。对于这些疾病，风险降低了大约10倍；这意味着，如果有两个保护性变体副本，10个患者中就有9个不会患上这种疾病。保护性变异体不会增加患恶性肿瘤的风险，也不会导致免疫缺陷。我们将开发一种策略，允许我们改变骨髓来源干细胞的遗传序列，以模仿大规模遗传研究确定的保护性遗传变异。然后，这些经过编辑的细胞可以用来通过干细胞移植用转基因细胞重新填充免疫系统。这种方法可能会根除有害的免疫细胞，并显着和永久性地减少复发的机会。这是一种治疗IMD的全新方法，它可能适用于各种不同的疾病。通过我们的编辑策略，我们将改变定义蛋白质结构的DNA序列，以模仿自然产生的保护性变体，从而削弱其功能。这种蛋白在免疫细胞激活中起着关键作用，它将干净而准确地降低免疫反应。编辑策略将在模拟多发性硬化症病理的IMD小鼠模型中进行测试，以确定编辑是否可以防止IMD的发展。我们还将确定需要多大比例的编辑细胞才能发挥保护作用，方法是使用编辑细胞和正常细胞的混合物在小鼠身上进行移植。

Autologous haemopoietic stem cell transplantation (ASCT) is emerging as an important therapy for patients with severe immune-meditated diseases (IMDs). Recent studies have shown that it is safe and effective for treating severe IMDs such as multiple sclerosis (MS) and scleroderma. This involves collecting bone marrow derived stem cells from a patient and then eliciting severe but transient immunosuppression using a combination of chemotherapy and therapeutic antibodies. This eradicates a large portion of the immune system including the autoreactive cells. Bone marrow derived stem cells are then reinfused and the immune system is reconstituted afresh with minimal long-term side effects. European Bone Marrow Transplant registry data show that more than 2500 patients have had autologous transplants for IMDs. Only one death has occurred since 2005 following autologous transplantation for MS. ASCT is superior to all other therapies in MS for inducing long-term remissions and it is the only treatment that has been found to reduce disability. In scleroderma, ASCT results in a very marked improvement in survival (86% v 51% at 6 years). Although ASCT can halt disease progression for many patients, 30-50% eventually re-develop their original IMD and 5-10% develop a different IMD. There is therefore significant scope to improve this therapeutic strategy. In recent years there has been a revolution in molecular biology due to programmable nucleases such as CRISPR-Cas9 because they allow us to make precise changes to the sequence of the genome. They are likely to become hugely important therapeutic tools within the next decade. We are using this technology to modify the genome sequence in bone marrow derived stem cells for curing inherited disorders of blood cell production and the immune system. The next step is to use this approach to treat acquired disorders of the immune system such as IMDs. Large-scale genetic analyses have revealed a target gene that is broadly protective across 20 different IMDs including multiple sclerosis (MS), scleroderma, rheumatoid arthritis and Crohn's disease. For these diseases there is around a 10- fold risk reduction; meaning that 9 in 10 patients would not have developed the disease had they had two copies of the protective variant. The protective variant does not result in increased risk for malignancy and does not lead to immunodeficiency. We will develop a strategy that allows us to change the genetic sequence of bone marrow-derived stem cells to mimic the protective genetic variants identified by large-scale genetic studies. These edited cells could then be used to repopulate the immune system with genetically modified cells with a stem cell transplant. This approach would potentially eradicate the harmful immune cells and significantly and permanently reduce the chance of relapse. This is a completely novel approach for treating IMDs and it is potentially applicable to a broad range of different diseases. Through our editing strategy we will change the DNA sequence that defines the structure of the protein, to mimic the naturally occurring protective variants, which attenuate its function. This protein plays a critical role in immune cell activation and it will cleanly and precisely decrease the immune response. The editing strategy will be tested in an IMD mouse model, which mimics the pathology of MS, to determine if the edit protects against IMD development. We will also define what proportion of edited cells required for the protective effect by performing transplants in mice using mixtures of edited and normal cells.