In Vivo Gene Editing of B cells with NICE-AAV Vectors

摘要 针对HIV的基因治疗是实现HIV治愈/稳定缓解的有效和有前途的手段。 缺乏抗逆转录病毒治疗(ART)。广谱中和抗体(BNAbs)和类似分子，如 由于eCD4-Ig提供了最清晰的治愈途径之一，但受到三个关键障碍的阻碍。第一，被动 根据定义，给予bNAb/eCD4-Ig蛋白是一种短暂的治疗；当这些蛋白的循环水平 强大的抗艾滋病毒因子下降，病毒复制就能恢复。第二，基因治疗载体为主 包括腺相关病毒(AAV)在内的方法支持延长bNAbs的表达和其他抗病毒药物 转基因，但往往受到宿主免疫反应的限制。第三，有效的抗逆转录病毒疗法抑制病毒 复制到极低水平，使工程化的HIV特异性淋巴细胞无法识别和清除 持续感染的细胞。我们已经生成了一套令人兴奋的工具和初步数据，直接解决了 其中的每一个障碍。克服bNAbs的暂时性和相关的载体免疫原性 给药方法，我们在非人灵长类动物(NHP)中进行了活体筛查，并发现 长期存在的工程化AAV变种(与宿主免疫系统缺乏识别一致)， 并专门针对B细胞。B细胞亲和性载体将与CRISPR-Cas9基因编辑机一起包装， 应用高度创新的共价连接方法将我们载体的包装能力提高一倍。我们指的是 我们的新体内递送方法是非免疫原性的货物增强型(NICE)AAV：单剂，NICE- AAV载体将用针对天然免疫球蛋白基因的bNAb或eCD4-Ig序列来特异性地重新编程B细胞。 最后，我们将通过提供与细胞相关的艾滋病毒-1来克服病毒抗原不足的重大问题。 环境在运输中。我们最近在NHP模型中发表的文章展示了这一战略的巨大成功 刺激HIV-1特异性嵌合抗原受体(CAR)T细胞，同样应该促进和触发扩张 我们的基因编辑的B细胞。我们建议的中心目标是验证B的有效性和特异性 细胞靶向NICE-AAV(AIM 1)，以证明这种体内递送方法能够持续 BNab/eCD4-Ig在HIV解剖区段和储存区的表达(AIM 2)，最重要的是， 在人源化小鼠和艾滋病毒持续存在的NHP模型中实现治疗效果(AIM 3)。我们将合并 最有希望治愈HIV的治疗方法之一(bNAbs/eCD4-Ig)具有非常独特的 VIVO交付平台(NICE-AAV)。重要的是，这种方法不仅适用于艾滋病毒-1，而且适用于 广泛的病理学，其中单抗疗法提供临床益处。

ABSTRACT HIV-specific gene therapies are a powerful and promising means to achieve HIV cure/stable remission in the absence of antiretroviral therapy (ART). Broadly neutralizing antibodies (bNAbs) and analogous molecules such as eCD4-Ig offer one of the clearest paths to a cure, but are hindered by three key obstacles. First, passive administration of bNAb/eCD4-Ig proteins is by definition a transient therapy; when circulating levels of these potent anti-HIV factors decline, virus replication is able to resume. Second, gene therapy vector-based approaches including adeno-associated virus (AAV) support prolonged expression of bNAbs and other antiviral transgenes, but are frequently limited by host immune responses. Third, potent ART regimens suppress viral replication to extremely low levels, rendering engineered HIV-specific lymphocytes unable to recognize and clear persistently infected cells. We have generated an exciting set of tools and preliminary data that directly addresses each of these barriers. To overcome the transient nature of bNAbs and associated immunogenicity of vectored delivery approaches, we have performed an in vivo screen in nonhuman primates (NHP) and identified engineered AAV variants that persist long term (consistent with a lack of recognition by the host immune system), and specifically target B cells. B cell tropic vectors will be packaged with CRISPR-Cas9 gene editing machinery, applying highly innovative covalent linkage methodology to double our vectors’ packaging capacity. We refer to our novel in vivo delivery approach as Non-Immunogenic, Cargo-Enhanced (NICE) AAV: in a single dose, NICE- AAV vectors will specifically reprogram B cells with bNAb or eCD4-Ig sequences targeted to the native IgG locus. Finally, we will overcome the significant problem of insufficient viral antigen by supplying cell-associated HIV-1 Env in trans. Our recent publication in the NHP model demonstrates the immense success of this strategy to stimulate HIV-1-specific chimeric antigen receptor (CAR) T cells and should similarly boost and trigger expansion of our gene-edited B cells. The central goals of our proposal are to validate the efficiency and specificity of B cell-targeted NICE-AAV (AIM 1), to demonstrate that this in vivo delivery approach enables persistent bNAb/eCD4-Ig expression in HIV anatomical compartments and reservoir sites (AIM 2), and most importantly, to achieve a therapeutic impact in humanized mouse and NHP models of HIV persistence (AIM 3). We will merge one of the most promising therapeutic modalities for HIV cure (bNAbs/eCD4-Ig) with our extremely unique in vivo delivery platform (NICE-AAV). Importantly, this approach will be applicable not only for HIV-1, but for the broad range of pathologies where monoclonal antibody therapies offer clinical benefit.