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

摘要 私人实体和学术团体都在为治疗目的开创B细胞编辑， 表达来自其天然免疫球蛋白基因座的治疗性抗体，或来自 异位基因。BCR编辑目前使用CRISPR/CAS系统和同源- 定向修复模板。非bcr转基因是通过逆转录病毒转导或 基于转座子的随机插入。基于核酸酶和基于插入的B细胞工程 技术带有与染色体缺失(CRISPR/CA)或插入 突变(逆转录病毒/转座子)。此外，只有通过以下方式才能进行有效的编辑 在体外分离和编辑B细胞意味着基于这些编辑方案的治疗将 可能会很贵。 我们已经发现了一种不需要外源核酸酶的B细胞编辑方法，并且 不依赖随机插入。我们的方法依赖于用DNA转换类别转换的B细胞 重组腺相关病毒(RAAV)载体提供的模板。倒置的端子 RAAV中的重复(ITR)序列自然整合为由 B类单元机--开关机械。有了正确的表达盒设计，我们可以取代 内源性重链可变区(VH)片段甚至表达非抗体转基因 在bcr基因内。这种无核酸酶技术在安全性方面具有潜在的优势。 而且，因为它只需要一个rAAV转导事件，所以它还承诺了一个简单的成本 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，并为 治疗应用。在目标1中，我们将优化我们的rAAV交付维修的设计 模板，并与基于CRISPR/CA的方法相比，证明了我们方法的相对安全性 正在编辑。在目标2中，我们测试了抗体和非抗体的不同表达框设计 转基因表达，并确定是否包含具有顺式作用的遗传元件 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， 我们将解决体内编辑问题。我们将确定是否在体内进行无核酸酶编辑 在注射rAAV之前接种小鼠以驱动B细胞型可以提高效率 相对于疫苗接种前的步骤，切换和优化我们的rAAV剂量和时间。我们会 还展示了我们编辑的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.