Gene Editing in X-Linked Agammaglobulinaemia

x连锁无球蛋白血症（XLA）是一种遗传性免疫缺陷，每25万新生儿中就有1人患病。它是由布鲁顿酪氨酸激酶（Btk）的突变引起的，该突变被编码在X染色体上。这种分子在B细胞祖细胞中表达，并提供生存信号，使B细胞成熟，然后产生抵抗感染的免疫球蛋白。没有这种分子，患者就没有b细胞，也就不能产生免疫球蛋白。因此，它们抵抗病原体的能力较弱，从而获得严重感染。传统疗法包括终身输注免疫球蛋白，但累积费用很高，而且在一些发展中国家无法获得。最近，血浆捐献的短缺导致了免疫球蛋白的供应压力。即使采用这种疗法，患者仍可能经常感染，这可能导致慢性肺部疾病，即支气管扩张。当患者对治疗依从性差时，这种风险会增加。尽管有足够的免疫球蛋白替代治疗，仍可出现炎症性肠病、慢性贾第虫病、慢性鼻窦炎和复发性结膜炎等并发症。目前缺乏一种安全和确定的治疗方法。基因治疗的最新进展为包括SCID、Wiskott Aldrich综合征和CGD在内的许多免疫缺陷提供了原理证明。这一过程消除了移植物抗宿主病的风险，并可能降低与同种异体手术相关的总体风险。这项技术涉及使用逆转录病毒载体将受影响基因的“功能”版本转移到造血干细胞或祖细胞中。该基因在半随机的位置整合到宿主DNA中。虽然有效，但对于需要精确基因表达和调控的条件，它可能不是完全有效，并且可能存在与基因表达失调相关的其他问题。另一种新颖的方法是使用精确的基因编辑技术，以便在能够进行生理基因表达的位置插入治疗性转基因。该项目将是一项临床前可行性研究，以建立该技术治疗XLA。首先，永生细胞系将被编辑，但随着技术的完善，人类干细胞将使用来自正常供体和XLA患者的干细胞。专门的酶将被转移到细胞中，在BTK基因的某个区域切割DNA。随后，使用病毒载体将正确版本的基因传递到细胞中，并将其转移到切割区域。然后对编辑过的细胞进行DNA检查，以证明这些改变是在正确的地方进行的，而不是在其他地方（即所谓的“脱靶”效应）。随后，将对编辑过的细胞进行测试，以确保它们能够分化成产生免疫球蛋白的b细胞。我们的目标是在免疫缺陷小鼠中测试这些细胞，观察小鼠是否开始制造B细胞和免疫球蛋白。近年来，全基因组测序的成本大幅下降，现在更容易引出疾病的遗传原因，包括原发性免疫缺陷。基因编辑有可能逆转这些突变。如果应用于胚胎，它可以阻止遗传性疾病的遗传。然而，这种应用是有争议的，不太可能在不久的将来进行试验。尝试对患有单基因免疫缺陷的成年人进行基因编辑是将这项技术应用于临床实践的更现实的第一步。如果这个项目是成功的，那么它可以适用于其他免疫学和血液学条件。

X-Linked Agammaglobulineamia (XLA) is an inherited immunodeficiency affecting 1 in 250,000 births. It is caused by a mutation in Bruton's Tyrosine Kinase (Btk) which is encoded in the X chromosome. This molecule is expressed in B Cell progenitors and provides survival signals to allow maturation into functional B Cells which then produce immunoglobulins which fight infections. Without this molecule patients do not have B-Cells and are unable to produce immunoglobulins. As a result they are less able to fight pathogens and therefore acquire severe infections. Conventional therapy comprises life-long infusions of immunoglobulin but the cumulative cost is high and this is not available in some developing countries. Recently a shortage of plasma donations has led to immunoglobulin supply pressures. Even with this therapy patients may still have frequent infections and these can lead to a chronic lung disease known as bronchiectasis. This risk increases when patients are poorly compliant with treatment. Complications such as inflammatory bowel disease, chronic giardiasis, chronic sinusitis and recurrent conjunctivitis are seen despite adequate Immunoglobulin replacement therapy. Currently a safe and definitive treatment is lacking.Recent progress in gene therapy has provided proof of principle in a number of Immunedeficiencies including SCID, Wiskott Aldrich Syndrome and CGD. This process obviates the risk of graft versus host disease and probably reduces the overall risk associated with allogeneic procedures. This technology involves the use of a retroviral vector to transfer a 'functional' version of the affected gene into haematopoietic stem or progenitor cells. The gene is integrated into host DNA at a semi-random location. While effective, for conditions where accurate gene expression and regulation is desirable, it may not be fully effective, and there may be additional problems associated with dysregulated gene expression. An alternative novel approach is to use accurate gene editing technology in order to insert a therapeutic transgene in a location that enables physiological gene expression. This project will be a pre-clinical feasibility study to establish this technique for the treatment of XLA. At first, immortalised cell lines will be edited but as the technique is refined, human stem cells will be used from normal donors and XLA patients. Specialised enzymes will transferred into cells to cut the DNA at an area in the BTK gene. Subsequently a correct version of the gene will be delivered into cells using a viral vector and this will be transferred into the area of the cut. . The edited cells will then have their DNA checked to show that the changes have been made in the correct place and not elsewhere (so called "off target" effects). Subsequently, the edited cells will be tested to ensure they can differentiate into B-Cells which produce Immunoglobulin. We aim to then test these cells in immune deficient mice and observe whether the mice start to make B Cells and Immunoglobulins. In recent years the cost of whole genome sequencing has fallen greatly and now it is easier to elicit the genetic cause of diseases including Primary Immunodeficiencies. Gene editing has the potential to reverse these mutations. If applied to embryos it could stop hereditary disease being inherited. However, this application is controversial and is unlikely to be trialled in the near future. Attempting gene-editing in adults with monogenic immunodeficiencies is a more realistic first step to bring this technology to clinical practice. If this project is successful then it can be adapted for other immunological and haematological conditions.