Pre-Clinical Development of Nuclease Mediated Gene Therapy for SCID

描述(申请人提供)：严重联合免疫缺陷(SCID)是一组遗传性疾病，患者出生时带有单基因突变，无法形成功能性免疫系统。虽然骨髓移植可以治愈这些疾病，但这种方法仍然有很大的局限性。使用逆转录病毒载体的基因治疗已经用于SCID，结果喜忧参半。数十名患者已经发展出功能免疫系统，这样他们就可以生活下去，而不必担心发生危及生命的感染。然而，少数患者由于原癌基因的插入激活而患上了医源性白血病。在针对这些疾病的下一代基于病毒的基因治疗试验中，病毒载体已经被设计成降低激活原癌基因的可能性。这些试验的结果，特别是致癌风险，在几年内都不会公布。我们一直致力于第三代SCID的基因治疗方法。与使用病毒载体以不受控制的方式传递转基因不同，我们正致力于使用同源重组来精确修改基因组。我们已经证明，使用工程核酸酶，我们可以在细胞系和原代细胞中通过同源重组(“基因靶向”)刺激在治疗上有用的频率(&gt；10%)的基因修饰。该提案的总体重点是以一种有条不紊和谨慎的方式将发现转化为用于患者来源的脐带血CD34+细胞。这三个具体目标集中在将我们的基础科学研究转化为临床试验。具体目的1是发展核酸酶介导的针对IL2RG基因的基因靶向。具体目的2是发展核酸酶介导的针对ADA基因的基因靶向。具体目标3侧重于使用全基因组方法来更好地了解核酸酶的靶上活性和靶外效应。我们这项提议的目标是完成必要的IND使能实验，以启动使用同源重组治疗SCID的首例人类基因治疗临床试验。

DESCRIPTION (provided by applicant): The severe combined immunodeficiencies (SCID) are a set of genetic diseases in which patients are born with mutations in single genes and are unable to develop functional immune systems. While bone marrow transplantation can be curative for these diseases there remain significant limitations to this approach. Gene therapy using retroviral vectors have been used for SCID with mixed results. Tens of patients have developed functional immune systems such that they can live without having to worry about developing life-threatening infections. A handful of patients, however, have developed iatrogenic leukemia from the insertional activation of a proto-oncogene. In the next generation of viral based gene therapy trials for these diseases, the viral vectors have been engineered to decrease the probability of activating proto- oncogenes. The results of these trials, particularly the oncogenic risk, will not be known for several years. We have been working towards a third generation gene therapy approach to SCID. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards using homologous recombination to precisely modify the genome. We have shown that using engineered nucleases we can stimulate gene modification by homologous recombination ("gene targeting") at frequencies that should be therapeutically useful (>10%) in cell lines and primary cels. The overall focus of the proposal is to translate the findings in a methodical and careful fashion to use in patient-derived umbilical cord blood derived CD34+ cells. The three specific aims are focused on translating our basic science studies into a clinical trial. Specific aim 1 is focused o developing nuclease mediated gene targeting for the IL2RG gene. Specific aim 2 is focused on developing nuclease mediated gene targeting for the ADA gene. Specific aim 3 is focused on using whole genome approaches to better understand nuclease on-target activity and off-target effects. Our goal with this proposal is to complete the IND-enabling experiments necessary to initiate a first-in-man gene therapy clinical trial using homologous recombination to cure SCID.