VLVs as platform for nucleic acid-based delivery:Combining therapeutic vaccine with shRNA-mediated PD-L1 blockade for the treatment of chronic hepatitis B virus

VLV 作为核酸递送平台：将治疗性疫苗与 shRNA 介导的 PD-L1 阻断相结合，治疗慢性乙型肝炎病毒

• 批准号: 10006651
• 负责人: Valerian Nakaar
• 金额: $ 30万
• 依托单位: CAROGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-14 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006651
• 关键词: Address; Affect; Animals; Antibodies; Antigens; Antiviral Therapy; Apoptosis; Automobile Driving; Biotechnology; CD8-Positive T-Lymphocytes; Cell Line; Cell physiology; Cells; Chronic; Chronic Hepatitis B; Cirrhosis; Clinical; Combined Modality Therapy; Communicable Diseases; Data; Development; Development Plans; Disease; Engineering; Feedback; Foundations; Genetic Engineering; Goals; HIV; Health; Hepatitis B Core Antigen; Hepatitis B Surface Antigens; Hepatitis B Virus; Human; Human Papillomavirus; Immune checkpoint inhibitor; Immune response; Immunity; Immunotherapy; Individual; Kinetics; Legal patent; Ligands; Liver; Liver diseases; Malaria; Malignant Neoplasms; Mediating; Methods; Modeling; Mus; Nucleic Acids; Open Reading Frames; PD-1 blockade; Pathway interactions; Phase; Phase I Clinical Trials; Polymerase; Prevention; Primary carcinoma of the liver cells; Public Health; Publications; RNA; RNA Precursors; Refractory; Replicon; Research; Resolution; Risk; Serum; Small Interfering RNA; Speed; System; T cell response; T-Lymphocyte; Technology; Testing; Therapeutic; Tissues; Toxicology; Treatment Efficacy; Vaccine Therapy; Vaccines; Vesicle; Viral; Viremia; Virus; Virus Diseases; Woodchuck; Woodchuck Hepatitis B Virus; anti-PD-L1; artificial vesicle; base; chronic infection; chronic liver disease; clinical candidate; commercialization; design; entecavir; exhaust; immunogenic; improved; in vivo; innovation; mouse model; nanoparticle; next generation; novel strategies; novel therapeutics; novel vaccines; nucleic acid-based therapeutics; prevent; programmed cell death protein 1; programs; receptor; research and development; response; small hairpin RNA; therapeutic development; therapeutic vaccine; treatment strategy; vector; vector vaccine; viral RNA

PROJECT SUMMARY/ABSTRACT Nucleic acids (NA) have a broad and expanding therapeutic potential, but a major limiting factor remains the difficulty to deliver these molecules. We have developed a highly scalable platform technology with the capacity for delivering NAs for use in multiple disease systems. Our goal is to adapt this technology to deliver small interfering RNA (siRNA) precursor form, short-hairpin (shRNA) as an NA–based therapeutic. In proof-of- concept studies, we have established that a VLV (RNA replicon-based vector) carrying RNA encoding all three HBV major antigens (MHBs, HBcAg and polymerase) in a single ORF (VLV-3xT2A) drives a broad multi- specific immune response that resulted in substantial clearance of HBV in the liver. In order to achieve long term viral suppression or complete elimination of the virus in the liver, we have targeted for disruption the checkpoint inhibitor, programmed cell death receptor ligand 1 (PD-L1) by shRNA technology. The hypothesis is that disruption of the PD-1/PD-L1 pathway will reinvigorate the otherwise exhausted T cell function. In preliminary studies, we have engineered CARG-101 to incorporate one or multiple copies of shRNA against PD-L1, and we have showed that these vectors specifically downregulate stably transfected PD-L1 in a BHK21 cell line. In this study, we will examine whether the in vivo blockade of the PD-1/PD-L1 pathway will enhance virus-specific T cell immunity that will lead to the complete resolution of chronic infection in mice. To this end, we will carry out the following specific aims: (1) Optimize VLV-shRNA vectors for inhibition of PD-L1; (2) Investigate the kinetics of PD-L1/PD-L2 and PD-1 expression in naïve and chronic HBV mice; and (3)Evaluate clearance using the AAV-HBV mouse model of HBV persistence. At the completion of Phase I, we will have: (a) established that VLV platform can deliver NAs to cells and tissues in vivo; (b) generated novel therapeutic that enhances the efficacy of therapeutic vaccine. A robust CD8+ T cell response via PD-1/PD-L1 pathway blockade will accrue, thereby driving a drastic or complete clearance of HBV in the liver. The demonstrated capacity to express NAs has broad utility for treating multiple diseases. Another compelling value proposition of this technology lies in its potential application to chronic and intractable diseases such as HIV, HPV, TB, malaria, and cancer which are refractory to current vaccine technologies but which may now be amenable to VLV technology.

项目总结/摘要 核酸（NA）具有广泛和不断扩大的治疗潜力，但主要的限制因素仍然是 很难传递这些分子。我们开发了一种高度可扩展的平台技术， 提供NAs用于多种疾病系统的能力。我们的目标是利用这项技术 小干扰RNA（siRNA）前体形式，短发夹（shRNA）作为基于NA的治疗剂。在证明- 概念研究中，我们已经建立了一个VLV（RNA复制子为基础的载体）携带RNA编码所有三个 单个ORF（VLV-3xT 2A）中的HBV主要抗原（MHB、HBcAg和聚合酶）驱动广泛的多个 特异性免疫应答，导致肝脏中HBV的大量清除。为了实现长期 术语病毒抑制或完全消除肝脏中的病毒，我们针对破坏 检查点抑制剂，程序性细胞死亡受体配体1（PD-L1）通过shRNA技术。这个假设是 PD-1/PD-L1通路的破坏将重新激活原本已耗尽的T细胞功能。在 在初步研究中，我们已经改造了CARG-101，使其包含一个或多个拷贝的shRNA， PD-L1，并且我们已经表明这些载体特异性下调BHK 21细胞中稳定转染的PD-L1。 细胞系在这项研究中，我们将检查PD-1/PD-L1通路的体内阻断是否会增强 病毒特异性T细胞免疫，这将导致小鼠慢性感染的完全解决。为此目的， 我们将进行以下具体目标：（1）优化VLV-shRNA载体以抑制PD-L1;（2） 研究PD-L1/PD-L2和PD-1在初治和慢性HBV小鼠中表达的动力学;以及（3）评估 使用HBV持续存在的AAV-HBV小鼠模型测定清除率。在第一阶段完成后，我们将： (a)建立了VLV平台可以在体内将NA递送至细胞和组织;（B）产生了新治疗剂 增强治疗性疫苗的效力。通过PD-1/PD-L1途径的稳健CD 8 + T细胞应答 阻断将增加，从而驱动肝脏中HBV的急剧或完全清除。证明的 表达NAs的能力对于治疗多种疾病具有广泛的用途。另一个引人注目的价值主张 这项技术的潜在应用在于它对慢性和难治性疾病，如艾滋病毒，人乳头瘤病毒，结核病， 疟疾和癌症，这些疾病对目前的疫苗技术是难治的，但现在可能适用于 VLV技术。