In Vivo Gene Editing for HIV-1 Cure

体内基因编辑治疗 HIV-1

• 批准号: 10331787
• 负责人: IRVIN S.Y. CHEN
• 金额: $ 67.8万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-11 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331787
• 关键词: AIDS therapy; Ablation; Address; Anti-Retroviral Agents; Antibody Therapy; BLT mice; Bar Codes; Binding; Biodistribution; Bioinformatics; Biological Products; Blood Circulation; Brain; CCR5 gene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell surface; Cells; Charge; Chemical Engineering; Chemical Structure; Clinical Research; Complex; Crosslinker; DNA; Disease; Encapsulated; Engineering; Freeze Drying; Gene Delivery; Gene-Modified; Genes; Genetic Engineering; Genetic Translation; Guide RNA; HIV Genome; HIV-1; Hematopoietic; Hematopoietic stem cells; Homing; Immune; Immune system; In Situ; In Vitro; Individual; Infection; Intravenous; Journals; Knock-out; Lentivirus Vector; Ligands; Liver; Lung; Lymphoid; Mainstreaming; Modeling; Mutate; Nanotechnology; Nucleic Acids; Organ; Patients; Pharmaceutical Preparations; Polymers; Positioning Attribute; Property; Proteins; Proviruses; Publications; Reagent; Resistance; Rest; Role; Scientist; Site; Small Interfering RNA; Surface Properties; System; T-Lymphocyte; Testing; The Sun; Therapeutic; Thinness; Time; Tissues; Transduction Gene; Viral Vector; Virion; Work; clinical practice; delivery vehicle; endonuclease; experience; gene therapy; immunogenicity; in vivo; knock-down; macromolecule; macrophage; monomer; mouse model; nanocapsule; nanotechnology platform; neoplastic cell; novel; nucleic acid-based therapeutics; particle; polymerization; preclinical study; reactivation from latency; receptor; small molecule; tool; transcription activator-like effector nucleases; vector; virtual; zinc finger nuclease

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.

项目概要 该提案要测试的总体假设是一类新型纳米胶囊可以有效地传递 将基因编辑成分导入到两个主要的 HIV-1 靶细胞、T 细胞和巨噬细胞中，并进行诱变 HIV-1原病毒，使得复制和/或潜伏期的重新激活被中止。虽然基因修饰是 具有挑战性，相对于小分子药物的优势在于 HIV-1 原病毒或 HIV-1 必需的基因 表达和/或感染可以直接被敲低或敲除，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)、转录激活子样效应核酸酶 (TALEN)、归巢核酸内切酶以及大多数 最近，CRISPR/Cas9系统。尽管这些新的基因编辑工具前景广阔，但治疗性核酸 酸和蛋白质很快就会从循环中流失，并且运载工具无法传递基因修饰 试剂通过有效手段影响HIV-1储存库。因此，迄今为止，所有基因修饰的应用 HIV-1疾病目前是在从体内取出并离体转导的细胞上进行的。从我们的过去 凭借工程化慢病毒载体的经验，我们认识到开发慢病毒载体工具的艰巨挑战 体内基因编辑，也是将基因治疗带入主流临床实践的希望和潜力。 我们之前的经验告诉我们，病毒载体存在滴度有限、生物分布不够充分、传播效果差等问题。 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，单个大分子、蛋白质、siRNA、gRNA 或 DNA 通过单体的原位聚合封装和保护在薄聚合物壳内，并通过 环境响应型交联剂。在许多方面，这些“纳米胶囊”与病毒粒子相似， 大小相似，并且像病毒粒子一样，保护单个包裹的基因。然而，他们的优势在于 简单制造以获得更高的“滴度”，通过冻干储存，最重要的是，能够轻松改变 化学结构、电荷和配体共轭的表面特性决定了以下因素 生物分布、细胞结合和进入。由于纳米胶囊的特性是由壳赋予的， 屏蔽货物，几乎任何核酸或蛋白质货物都可以互换。通过明智的选择 聚合物壳和交联剂，我们成功地设计了纳米胶囊，增强了生物分布 储库位点，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，这些纳米胶囊本身相对非 具有免疫原性并保护货物免受免疫系统的影响。这些原理证明研究开始 克服上述挑战，从而为我们提出的研究奠定基础。