In Vivo Gene Editing for HIV-1 Cure

体内基因编辑治疗 HIV-1

• 批准号: 9753575
• 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/9753575
• 关键词: AIDS therapy; Ablation; Address; Anti-Retroviral Agents; Antibody Therapy; BLT mice; Bar Codes; Binding; Biodistribution; Bioinformatics; Biological; 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; 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.

项目摘要 该提案中要检验的总体假设是，新型的纳米胶囊可以有效地提供 基因编辑成分进入两个主要的HIV-1靶细胞，T细胞和巨噬细胞，并诱变 HIV-1病毒的复制和/或延迟重新激活被中止。而基因修饰是 具有挑战性的是，对小分子药物的优势是HIV-1病毒或HIV-1所需的基因 表达和/或感染可以直接击倒或击倒，而无需杀死感染 细胞。包括锌指（包括锌指）的许多系统已经实现了有效的基因修饰活性 核（ZNF），转录激活剂样效应核（Talens），归巢核酸内切酶和大多数 最近，CRISPR/CAS9系统。尽管有这些新基因编辑工具的承诺，但治疗核 酸和蛋白质因循环而迅速损失，输送车辆无法传递基因修饰 通过有效影响HIV-1储层的试剂。迄今为止，基因修饰的所有应用 HIV-1疾病目前是在从体内去除并翻译过的细胞上实践的。从我们的过去 具有工程慢病毒载体的经验，我们认识到开发工具的困难挑战 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。 我们先前的经验告诉我们，病毒载体受到滴度的局限性，适当的生物分布，差 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是 通过原位聚合单体的原位聚合并稳定 环境响应迅速的交联。在许多方面，这些“纳米胶囊”类似于病毒粒子颗粒， 大小相似，并且像病毒一样保护单个包裹的基因。但是，它们的优势 简单制造到更高的“滴定”，通过冻干储存，最重要的是，可以轻松改变 化学结构，电荷和配体共轭的表面特性，这决定了因素 生物分布，细胞结合和进入。由于纳米胶囊的特性由外壳赋予 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 聚合物外壳和交联，我们成功地设计了纳米胶囊，从而增强生物分布 储层站点，以时间释放方式释放模型货物，并通过配体靶向特定的细胞 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，并将货物免受免疫系统的影响。这些原则研究的证据开始 克服上面概述的挑战，从而为我们提出的研究提供了基础。