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.

项目摘要/摘要 核酸（NA）具有广泛而扩大的治疗潜力，但主要的限制因素仍然是 很难传递这些分子。我们已经开发了一种高度可扩展的平台技术 用于在多种疾病系统中使用NAS的能力。我们的目标是调整这项技术以交付 小型干扰RNA（siRNA）前体形式，短发（shRNA）作为基于NA的治疗。在证明 概念研究，我们已经确定，携带RNA的VLV（基于RNA副本的矢量）编码这三个 单个ORF（VLV-3XT2A）中的HBV主要抗原（MHB，HBCAG和聚合酶）驱动宽阔的多种多样 特定的免疫反应导致肝脏中HBV的大量清除。为了长时间实现 术语病毒抑制或完全消除肝脏病毒，我们针对破坏了 检查点抑制剂，SHRNA技术的编程细胞死亡受体配体（PD-L1）。该假设是 PD-1/PD-L1途径的破坏将振兴原本耗尽的T细胞功能。在 初步研究，我们已经设计了Carg-101，以合并一个或多个shRNA反对 PD-L1，我们已经表明，这些向量在BHK21中特别下调了稳定翻译的PD-L1 细胞系。在这项研究中，我们将检查PD-1/PD-L1途径的体内封锁是否会增强 病毒特异性T细胞免疫将导致小鼠慢性感染的完全分辨率。为此， 我们将执行以下特定目标：（1）优化VLV-shRNA矢量以抑制Pd-L1； （2） 研究幼稚和慢性HBV小鼠中PD-L1/PD-L2和PD-1表达的动力学； （3）评估 使用HBV持久性的AAV-HBV鼠标模型进行清除。在第一阶段完成时，我们将有： （a）确定VLV平台可以在体内向细胞和组织提供NAS； （b）产生的新疗法 这提高了热真空的效率。通过PD-1/PD-L1途径强大的CD8+ T细胞响应 封锁将产生，从而驱动肝脏中HBV的严重或完全清除。证明了 表达NAS的能力具有广泛的治疗多种疾病的效用。另一个引人入胜的价值提议 这项技术在于它的潜在应用于慢性和顽固疾病，例如HIV，HPV，TB， 疟疾和对当前疫苗技术难治性的癌症，但现在可能适合 VLV技术。