Optimizing Gene Editing in Primary Human B Cells for Therapy and Research

摘要 特定的酶。目前治疗酵母菌病的方法是不够的，依赖于 骨髓移植或终生酶替代疗法。酶替代疗法可以 每年花费数十万美元，骨髓移植非常不稳定， 其中一个子集导致移植物抗宿主病死亡。另一种方法是 修改患者更具延展性和可访问性的细胞，如淋巴细胞，以表达野生型 并将这些细胞重新注入患者体内，以产生缺乏的 酵素。最近，人们在人类T细胞基因组工程方面做了大量的工作， 主要用于癌症免疫疗法。然而，长寿的T细胞亚群很大程度上 代谢不活跃，不适合持续的蛋白质生产。相反，B细胞可以产生 大量的保护性抗体，并持续多年，主要是由于 长寿的浆细胞。已经证明，这些浆细胞不仅仅是重新播种 通过记忆B细胞，而不是成为长寿抗体产生细胞的结果 而不是扩散。 产生大量蛋白质(即抗体)使我们假设这些细胞可能是 酶病基因治疗的理想平台。这导致我们调查其他人是否有 尝试使用靶向核酸酶修饰B细胞，令我们惊讶的是，我们没有发现任何文献 任何靶向核酸酶在原代人类B细胞中的应用。因此，我们进行了初步的 使用CRISPR/Cas9系统诱导B细胞双链断裂(DSB)的研究发现 我们可以以合理的效率编辑原代人类B基因，通过测量者核酸酶高达43% 化验。我们还定性地证明了我们可以将基因传递给B细胞 DSB诱导促进同源重组。在这里，我们提出了：1)优化基因 使用CRISPR/Cas9系统编辑并提供给原代人类B细胞，以及2)执行校样- 利用基因编辑的B细胞治疗酶病的概念研究。具体地说，我们将尝试 在NOD/SCID/IL2Rγ背景下治疗I型粘多糖病小鼠模型 表达已知抗原特异性bcr的工程化人B细胞移植 转录上与α-L艾杜糖酸酶(IDUA)连锁，随后进行针对 转基因bcr以产生长寿的浆细胞。

ABSTRACT Enzymopathies are a disturbance of enzyme function, including genetic deficiency or defect in specific enzymes. Current methods for the treatment of enzymopathies are insufficient and rely on bone marrow transplant or life long enzyme replacement therapy. Enzyme replacement therapies can cost hundreds of thousands of dollars per year and bone marrow transplant are highly precarious, with a subset resulting in death form graft versus host disease. An alternative approach would be to modify a patients more malleable and accessible cells, such as lymphocytes, to express a wild type version of the corrupted enzyme and re-infuse these cells into the patient to produce the lacking enzyme. Recently, there has been a great amount of work on genome engineering of human T cells, largely for cancer immunotherapies. However, the subsets of T cells that are long-lived are largely metabolically inactive and not ideal for constant protein production. Conversely, B cells can generate large amounts of protective antibodies and continue to do so for years, largely due to the activity of long-lived plasma cells. It has been demonstrated that these plasma cells are not merely re-seeded by memory B cells but instead are the result of becoming long-lived antibody producing cells that do not proliferate. The fact that B cells can become long lived and inherently have the metabolic activity to generate large quantities of protein (i.e. antibody) led us to hypothesize that these cells might be an ideal platform for gene therapy for enzymopathies. This led us to investigate if others had attempted to modify B cells using targeted nucleases and to our surprise we found zero publications on the use of any targeted nuclease in primary human B cells. Thus, we performed preliminary studies using the CRISPR/Cas9 system to induce double strand breaks (DSBs) in B cells and found that we can gene edit primary human B with reasonable efficiencies, up to 43% by Surveyor nuclease assay. We have also qualitatively demonstrated that we can deliver genes to B cells using homologous recombination enhanced by DSB induction. Here, we propose to: 1) Optimize gene editing and delivery to primary human B cells using the CRISPR/Cas9 system, and 2) Perform proof- of-concept studies to treat the enzymopathies using gene edited B cells. Specifically, we will attempt to treat a mouse model of Mucopolysaccharidosis type I on a NOD/SCID/Il2rγ background by transplantation of engineered human B cells expressing a BCR of known antigen specificity transcriptionally linked to Alpha-L-iduronidase (IDUA) with subsequent immunization specific to the transgene BCR to generate long lived plasma cells.