In Vivo Gene Editing for HIV-1 Cure

项目总结 这项提议要检验的总体假设是，一种新型的纳米胶囊可以有效地传递 基因编辑组件进入HIV-1的两个主要靶细胞，T细胞和巨噬细胞，并诱变 HIV-1前病毒使得从潜伏期复制和/或重新激活被中止。而基因改造则是 具有挑战性的是，与小分子药物相比，优势在于HIV-1前病毒或HIV-1所必需的基因 表达和/或感染可以被直接击倒或敲除，而不需要杀死感染者 细胞。包括锌指在内的许多系统已经实现了有效的基因修饰活性。 核酸酶(ZNF)、转录激活物样效应核酸酶(TALEN)、归巢内切酶和MOST 最近，CRISPR/CAS9系统。尽管这些新的基因编辑工具前景看好，但治疗性核 酸和蛋白质从循环中迅速流失，运送工具无法运送基因修饰 通过有效的手段来影响试剂对HIV-1宿主的影响。因此，到目前为止，所有用于基因改造的应用 HIV-1疾病目前是在从体内取出的细胞上进行的，并在体外进行转导。从我们的过去 在工程慢病毒载体方面的经验，我们认识到开发工具用于 活体基因编辑，也是将基因治疗带入主流临床实践的前景和潜力。 我们以前的经验告诉我们，病毒载体在滴度、适当的生物分布、糟糕的 静息T细胞转导，复合基因工程，免疫原性。最近，我们开发了一种 纳米技术平台，其中单个大分子、蛋白质、siRNA、gRNA或DNA 通过单体的原位聚合在薄的聚合物外壳中进行封装和保护，并通过 环保型交联剂。在许多方面，这些“纳米胶囊”与病毒粒子相似， 具有相似的大小，并且像病毒粒子一样，保护被包裹的单个基因。然而，它们的优势在于 制造简单，效价更高，通过冻干储存，最重要的是，能够轻松地改变 化学结构、电荷和配基共轭的表面性质决定了诸如 生物分布、细胞结合和进入。由于纳米胶囊的性质是由壳赋予的， 屏蔽货物，几乎任何核酸或蛋白质货物都可以互换。通过明智地选择 聚合物壳和交联剂，我们成功地设计出纳米胶囊，增强了生物分布 储藏部位，以时间释放方式释放模型货物，并通过配体靶向体内特定细胞 细胞表面分子的识别。此外，这些纳米胶囊本身是相对非 免疫原性，并保护货物免受免疫系统的侵害。这些原则性证明研究开始 克服上述挑战，从而为我们拟议的研究提供基础。

PROJECT SUMMARY The overall hypothesis to be tested in this proposal is that a novel class of nanocapsules can effectively deliver gene editing components into the two primary HIV-1 target cells, T-cells and macrophages, and mutagenize the HIV-1 provirus such that replication and/or reactivation from latency is aborted. While gene modification is challenging, the advantage over small molecule drugs is that the HIV-1 provirus or genes necessary for HIV-1 expression and/or infection can be directly knocked down or knocked out without the need to kill the infected cells. Efficient gene-modification activity has been achieved by a number of systems including zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), homing endonucleases, and most recently, the CRISPR/Cas9 system. Despite the promise of these new gene editing tools, therapeutic nucleic acids and proteins are rapidly lost from circulation and delivery vehicles cannot deliver gene modifying reagents by effective means to impact HIV-1 reservoirs. Thus, to date, all applications of gene modification for HIV-1 disease are currently practiced on cells removed from the body and transduced ex vivo. From our past experience with engineered lentiviral vectors, we recognize the difficult challenges of developing tools for in vivo gene editing, but also the promise and potential of bringing gene therapy into mainstream clinical practice. Our prior experience teaches us that viral vectors suffer from limitations in titer, adequate biodistribution, poor transduction of resting T-cells, complex genetic engineering, and immunogenicity. Recently, we developed a nanotechnology platform whereby individual macromolecules, protein, siRNA, gRNA, or DNA, are encapsulated and protected within a thin polymer shell by in situ polymerization of monomers and stabilized by environmentally responsive crosslinkers. In many respects, these “nanocapsules” are similar to virion particles, being of similar size and, like virions, protect the single encased gene. However, they have the advantage of simple manufacturing to higher “titer”, storage by freeze-dry, and, most importantly, the ability to easily alter the surface properties of chemical structure, charge, and ligand conjugation which determines factors such as biodistribution, cell binding, and entry. Since the properties of the nanocapsule are conferred by the shell which shields the cargo, virtually any nucleic acid or protein cargo can be interchanged. By judicious choice of polymer shell and crosslinkers, we successfully engineered nanocapsules which enhance biodistribution to reservoir sites, release a model cargo in time release fashion, and target specific cells in vivo through ligand recognition of cell surface molecules. Furthermore, these nanocapsules themselves are relatively non- immunogenic and shield the cargo from the immune system. These proof of principle studies begin to overcome the challenges outlined above and thus provide the basis for our proposed studies.