Safe, CRISPR/Cas-free B cell editing for therapeutic applications

摘要 私人实体和学术团体都在开拓用于治疗目的的B细胞编辑， 或者从它们的天然IG基因座表达治疗性抗体，或者从免疫球蛋白基因座表达其它转基因。 异位基因座BCR编辑目前使用CRISPR/Cas系统和同源- 定向修复模板。非BCR转基因通过逆转录病毒转导或 转座子随机插入基于核酸酶和基于插入的B细胞工程 技术存在与染色体缺失（CRISPR/Cas）或插入相关的风险 诱变（逆转录病毒/转座子）。此外，只有通过以下方式才能进行有效编辑 离体分离和编辑B细胞意味着基于这些编辑方案的治疗将 可能会非常昂贵。 我们已经发现了一种B细胞编辑的方法，该方法不需要外源核酸酶， 不依赖于随机插入。我们的方法依赖于用DNA转导类别转换的B细胞 由重组腺相关病毒（rAAV）载体提供的模板。反向末端 rAAV中的ITR序列天然整合到由重组腺病毒产生的双链断裂中。 B细胞类别转换机制。有了正确的表达盒设计，我们可以取代 内源性重链可变（VH）区段或甚至表达来自 在BCR基因座内。这种无核酸酶技术在安全性方面具有潜在优势 而且，因为它只需要一个单一的rAAV转导事件，它也承诺一个简单的，成本 体内编辑B细胞的有效手段。 在这里，我们的目标是开发我们的无核酸酶编辑技术，并提供概念验证， 治疗应用。在目标1中，我们将优化rAAV递送修复的设计 模板，并证明我们的方法与基于CRISPR/Cas的方法相比的相对安全性。 编辑.在目标2中，我们测试了抗体和非抗体的不同表达盒设计 转基因表达，并确定是否包含顺式作用遗传元件， 增加体细胞超突变可以增强我们编辑的B细胞的亲和力成熟。在目标3中， 我们将讨论体内编辑。我们将确定体内无核酸酶编辑是否 通过在rAAV施用之前接种小鼠以驱动B细胞类 切换和优化我们的rAAV剂量和相对于接种前步骤的时间。我们将 也证明了我们编辑的B细胞系统产生重组抗体（BCR）的能力 编辑）和促红细胞生成素（非抗体转基因表达）。

ABSTRACT Both private entities and academic groups are pioneering B cell editing for therapeutic purposes, either to express therapeutic antibodies from their native Ig loci, or other transgenes from an ectopic locus. BCR editing is currently performed with CRISPR/Cas system and a homology- directed repair template. Non-BCR trasngenes are introduced by retroviral transduction or transposon-based random insertion. Both nuclease-based and insertion-based B cell engineering techniques carry risks associated with chromosomal deletion (CRISPR/Cas) or insertional mutagenesis (retroviruses/transposons). Furthermore, efficient editing is only possible by isolating and editing B cells ex vivo meaning that therapies based on these editing protocols will likely be very expensive. We have discovered a method of B cell editing that requires no exogenous nucleases and does not rely on random insertion. Our method relies on transducing class-switching B cells with a DNA template supplied by a recombinant adeno-associated virus (rAAV) vector. The inverted terminal repeat (ITR) sequences in the rAAV naturally integrate into double-strand breaks created by the B cell class-switch machinery. With the right expression cassette designs, we can replace the endogenous heavy chain variable (VH) segment or even express a non-antibody transgene from within the BCR locus. This nuclease-free technique has potential advantages in terms of safety and, because it requires only a single rAAV transduction event, it also promises a simple, cost effective means of editing B cells in vivo. Here we aim to develop our nuclease-free editing technique and provide proof-of-concept for therapeutic applications. In Aim 1, we will optimize the design of our rAAV-delivered repair template, and demonstrate the relative safety of our approach compared to CRISPR/Cas-based editing. In Aim 2, we test different expression cassette designs for antibody and non-antibody transgene expression and determine whether or not inclusion of cis-acting genetic elements that increase somatic hypermutation can enhance affinity maturation of our edited B cells. In Aim 3, we will address in vivo editing. We will determine whether or not in vivo nuclease-free editing efficiency can be enhanced by vaccinating mice prior to rAAV administration to drive B cell class switching and optimize our rAAV doses and timing relative to the pre-vaccination step. We will also demonstrate the ability of our edited B cell system to produce recombinant antibodies (BCR editing) and erythropoietin (non-antibody transgene expression) in mice.