Pre-Clinical Development of Nuclease Mediated Gene Therapy for SCID

描述（由申请人提供）：严重联合免疫缺陷（SCID）是一组遗传性疾病，其中患者出生时具有单个基因突变，无法发育功能性免疫系统。虽然骨髓移植可以治愈这些疾病，但这种方法仍然存在明显的局限性。使用逆转录病毒载体的基因疗法已用于SCID，结果好坏参半。数十名患者已经发展出功能性免疫系统，因此他们可以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发生医源性白血病。在针对这些疾病的下一代基于病毒的基因治疗试验中，病毒载体已经被工程化以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，在几年内不会知道。我们一直致力于第三代基因治疗方法的SCID。与使用病毒载体以不受控制的方式传递转基因相反，我们正在努力使用同源重组来精确地修饰基因组。我们已经表明，使用工程化的核酸酶，我们可以在细胞系和原代大肠杆菌中以应该在治疗上有用（>10%）的频率通过同源重组（“基因靶向”）刺激基因修饰。该提案的总体重点是以有条不紊和谨慎的方式将研究结果转化为用于患者来源的脐带血来源的CD34+细胞。这三个具体目标的重点是将我们的基础科学研究转化为临床试验。具体目标1集中于开发针对IL 2RG基因的核酸酶介导的基因靶向。具体目标2集中于开发针对ADA基因的核酸酶介导的基因靶向。具体目标3集中于使用全基因组方法来更好地理解核酸酶的中靶活性和脱靶效应。我们的目标是完成IND启动实验，启动首次使用同源重组治疗SCID的人体基因治疗临床试验。

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.