Intraganglionic Analgesic Adeno-Associated Virus (AAV) Gene Vector Optimization in Large Animals

大型动物节内镇痛腺相关病毒 (AAV) 基因载体优化

• 批准号: 10021475
• 负责人: ANDREAS S. BEUTLER
• 金额: $ 68.76万
• 依托单位: INTERVENTIONAL ANALGESIX, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-14 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021475
• 关键词: Abnormal Laboratory Test Result; Acute; Address; Advanced Malignant Neoplasm; Adverse effects; Affect; American; Analgesics; Anatomy; Animal Model; Animal Testing; Animals; Bar Codes; Behavior; Biodistribution; Blood; Brain; Brain Stem; Capsid; Cartilage; Cells; Characteristics; Chronic; Clinical; Complex; Convection; DNA; Data; Degenerative polyarthritis; Dependovirus; Development; Distant; Dose; Drug Kinetics; Edema; Engineering; Epidemic; Esthesia; Family suidae; Financial cost; Fluoroscopy; Future; Gait; Gene Delivery; Gene Targeting; Gene Transduction Agent; Gene Transfer; Genes; Goals; Hallucinations; Health; Heroin; Histopathology; Human; Hyperalgesia; Image; Imaging technology; Immune response; Inflammation; Investments; Kinetics; Knee; Knee Osteoarthritis; Lead; Libraries; Liver; Magnetic Resonance Imaging; Methodology; Methods; Modeling; Modification; Morphologic artifacts; Mus; Needles; Neuraxis; Neurons; Nociception; Opioid; Opioid Analgesics; Opioid Peptide; Oral; Organ; Outcome Measure; Oxycodone; Pain; Pain management; Peripheral Nerves; Pharmaceutical Preparations; Pharmacology and Toxicology; Population; Primates; Protein Isoforms; Public Health; RNA; Rattus; Regimen; Reporting; Research; Resolution; Rodent Model; Serotyping; Severities; Spinal Cord; Spinal Ganglia; Structure; Syndrome; Techniques; Technology; Testing; Tissues; Toxic effect; Toxicology; Validation; Variant; Vertebral column; X-Ray Computed Tomography; addiction; adeno-associated viral vector; behavior test; beta-Endorphin; chronic pain; comparative; conventional therapy; cost; design; efficacy testing; experimental study; gene therapy; genotoxicity; head-to-head comparison; image guided; in vivo; mesolimbic system; opioid use; osteoarthritis pain; pain patient; pain signal; parity; phantom model; prescription opioid; promoter; relating to nervous system; respiratory; screening; secondary outcome; sex; side effect; synergism; therapeutic gene; transduction efficiency; transgene expression; vector; vector genome; viral RNA

Adeno-Associated Virus (AAV) gene therapy has the potential—as yet unrealized—to provide targeted anal- gesic agent delivery in pain patients, if optimized vectors can achieve a key objective in large animals: efficient and anatomically selective gene delivery to the neurons of a dorsal root ganglion (DRG). Such gene targeting could turn even opioids (gene-encoded) into a safe and non-addictive treatment of chronic locoregional pain. Pain is a national health problem affecting 116M Americans at a cost of $560B/year. Untargeted opioids used to treat pain such as oral oxycodone act on multiple neural structures other than the DRG causing side effects such as respiratory arrest (via the brainstem), hallucinations (via the cortex), and addiction (via the mesolimbic system). 18,893 Americans died from prescription opioids in 2014 and 1.9 million were addicted to them. Key technologies required for AAV gene therapy of pain already exist such as multiple therapeutic genes and a clear definition of a target cell population, DRG neurons. In fact, analgesic efficacy of DRG-directed AAV gene vectors has been thoroughly proven in rodent models. Astonishingly, however, large animal testing has not been reported for any analgesic gene vector, neither AAV nor any other vector type. Hypothesis: Anatomically selective delivery of AAV vectors encoding a known analgesic gene to select DRG can reverse pain-related behavior in a large animal model of a clinically common loco-regional pain syndrome. Aim 1. Optimize AAV vector design by high-throughput testing of strains in vivo in DRG of swine. A critical bottleneck in vector optimization is comparative testing of multiple strains in vivo. We will use the newly developed high throughput RNA-and-DNA “Barcode” sequencing methodology developed by the Co-I that allows concurrent quantification of transduction efficiency and transgene expression of up to hundreds of AAV strains in a single experiment to define the optimal vector characteristics for DRG neuron targeting. Aim 2. Develop Magnetic Resonance Imaging (MRI)-guided vector targeting to DRG in swine. MRI imaging of the swine spine showed superior demarcation of DRG compared with CT. MRI- mislocalization artifacts of needles used could be corrected in phantom models. We will develop MRI-guided AAV targeting of DRG by convection enhanced delivery while integrated toxicological endpoints into the aim. Aim 3. Test the analgesic efficacy of AAV in a model of chronic knee osteoarthritis (OA) pain in swine. Knee OA ranks amongst the two most common causes of chronic pain in humans. We established a model of chronic knee (“stifle”) OA in swine. We will use the model to test the analgesic efficacy and integrated toxicity endpoint of AAV expressing opioid genes in the DRG . Impact: Large animal gene transfer optimization and efficacy testing performed along with an assessment of toxicological endpoints will advance the concept of targeted analgesic AAV gene therapy of DRG to the point where a pre-IND discussion can be initiated and the additional investment in formal toxicology testing justified.

腺相关病毒（AAV）基因治疗有潜力（尚未实现）提供靶向分析 如果优化的载体可以在大型动物中实现关键目标：高效，则可以将 gesic 药物递送到疼痛患者中 以及解剖学上选择性的基因传递到背根神经节（DRG）的神经元。这种基因靶向 甚至可以将阿片类药物（基因编码）变成一种安全且非成瘾的慢性局部疼痛治疗方法。 疼痛是一个全国性的健康问题，影响着 1.16 亿美国人，每年造成的损失高达 560B 美元。使用非靶向阿片类药物 治疗疼痛，例如口服羟考酮作用于 DRG 以外的多个神经结构，导致副作用 例如呼吸停止（通过脑干）、幻觉（通过皮质）和成瘾（通过中脑边缘） 系统）。 2014 年，18,893 名美国人死于处方阿片类药物，190 万人对这些药物上瘾。 AAV 疼痛基因治疗所需的关键技术已经存在，例如多种治疗基因和 目标细胞群 DRG 神经元的明确定义。事实上，DRG 导向的 AAV 具有镇痛功效 基因载体已在啮齿动物模型中得到彻底证明。然而令人惊讶的是，大型动物试验已经 尚未报道任何镇痛基因载体，无论是 AAV 还是任何其他载体类型。 假设：编码已知镇痛基因的 AAV 载体在解剖学上选择性递送以选择 DRG 可以逆转临床常见局部疼痛综合征的大型动物模型中与疼痛相关的行为。 目标 1. 通过猪 DRG 体内菌株的高通量测试来优化 AAV 载体设计。 载体优化的一个关键瓶颈是体内多个菌株的比较测试。我们将使用 Co-I 开发的新开发的高通量 RNA 和 DNA“条形码”测序方法 允许同时量化多达数百个的转导效率和转基因表达 AAV 菌株在单个实验中定义了 DRG 神经元靶向的最佳载体特征。 目标 2. 开发针对猪 DRG 的磁共振成像 (MRI) 引导载体。 与 CT 相比，猪脊柱的 MRI 成像显示 DRG 的划分更清晰。 MRI- 所用针的错误定位伪影可以在模型模型中得到纠正。我们将开发 MRI 引导 AAV 通过对流增强递送以 DRG 为目标，同时将毒理学终点纳入目标。 目标 3. 在猪慢性膝骨关节炎 (OA) 疼痛模型中测试 AAV 的镇痛效果。 膝关节骨关节炎是人类慢性疼痛的两个最常见原因之一。我们建立了一个模型 猪的慢性膝关节（“膝关节”）OA。我们将使用该模型来测试镇痛功效和综合毒性 AAV 在 DRG 中表达阿片类基因的终点。 影响：大型动物基因转移优化和功效测试以及评估 毒理学终点将DRG靶向镇痛AAV基因治疗的概念推进到这一点 可以启动 IND 前讨论，并证明对正式毒理学测试的额外投资是合理的。