In Vivo Gene Editing for HIV-1 Cure

项目摘要 在这项提议中要测试的总体假设是，一类新型纳米胶囊可以有效地递送 将基因编辑组分导入两种主要的HIV-1靶细胞，T细胞和巨噬细胞，并诱变 HIV-1前病毒，从而使复制和/或从潜伏期的再激活中止。虽然基因改造是 具有挑战性的是，与小分子药物相比，HIV-1前病毒或HIV-1所需的基因 表达和/或感染可以被直接敲低或敲除，而不需要杀死感染者 细胞包括锌指蛋白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）、转录激活因子样效应物核酸酶（TALEN）、归巢核酸内切酶和大多数核酸内切酶。 CRISPR/Cas9系统。尽管这些新的基因编辑工具有希望， 酸和蛋白质从循环中迅速丢失，并且递送载体不能递送基因修饰 试剂通过有效手段影响HIV-1宿主。因此，到目前为止，基因修饰的所有应用， 目前，HIV-1疾病是在从体内取出并体外转导的细胞上进行的。从过去的 基于我们在工程慢病毒载体方面的经验，我们认识到开发用于治疗的工具的困难挑战。 体内基因编辑，也是将基因治疗纳入主流临床实践的前景和潜力。 我们先前的经验告诉我们，病毒载体在滴度、足够的生物分布、差的生物学特性、以及在细胞内的生物学特性方面受到限制。 静息T细胞的转导、复杂的基因工程和免疫原性。最近，我们开发了一个 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将其封装并保护在薄的聚合物壳内，并通过 环境响应性交联剂。在许多方面，这些“纳米胶囊”类似于病毒粒子， 它们大小相似，像病毒体一样，保护单个被包裹的基因。然而，它们具有以下优点： 简单的制造，以更高的“滴度”，冷冻干燥储存，最重要的是，能够容易地改变 化学结构、电荷和配体共轭的表面性质，其决定因素如 生物分布、细胞结合和进入。由于纳米胶囊的性质是由 屏蔽货物，实际上任何核酸或蛋白质货物可以互换。通过明智选择 聚合物壳和交联剂，我们成功地设计了纳米胶囊， 储库位点，以时间释放方式释放模型货物，并通过配体在体内靶向特定细胞， 识别细胞表面分子。此外，这些纳米胶囊本身是相对非- 免疫原性和屏蔽货物免疫系统。这些原理证明研究开始， 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.