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: 9445987
• 负责人: ANDREAS S. BEUTLER
• 金额: $ 66.61万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9445987
• 关键词: Abnormal Laboratory Test Result; Acute; Address; Advanced Malignant Neoplasm; Adverse effects; Affect; American; Analgesics; Anatomy; Animal Model; Animal Testing; Animals; 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; Patients; Peptides; Peripheral Nerves; Pharmaceutical Preparations; Pharmacology and Toxicology; Population; Primates; Protein Isoforms; Public Health; RNA; Rattus; Regimen; Reporting; Research; Resolution; Rodent Model; Serotyping; Severities; Signal Transduction; 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; opioid use; parity; phantom model; prescription opioid; promoter; relating to nervous system; respiratory; screening; secondary outcome; sex; 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）基因治疗有可能-尚未实现-提供靶向的肛门， 如果优化的载体可以在大型动物中实现关键目标，那么疼痛患者中的gesic药剂递送： 以及解剖学上选择性地将基因递送至背根神经节（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）变成一种安全且不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，每年花费5600亿美元。使用的非靶向阿片类药物 例如口服羟考酮作用于DRG以外的多个神经结构 例如呼吸停止（通过脑干）、幻觉（通过皮层）和成瘾（通过中脑边缘系统）。 系统）。2014年，18，893名美国人死于处方阿片类药物，190万人对它们上瘾。 AAV基因治疗疼痛所需的关键技术已经存在，例如多个治疗基因， 一个明确的目标细胞群的定义，背根神经节神经元。事实上，DRG导向的AAV的镇痛功效 基因载体已经在啮齿动物模型中得到了充分的证明。然而，令人惊讶的是， 没有关于任何镇痛基因载体的报道，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载体的解剖学选择性递送以选择DRG 可以逆转临床常见的局部疼痛综合征的大型动物模型中的疼痛相关行为。 目标1.通过猪背根节中菌株的体内高通量测试来优化AAV载体设计。 载体优化中的关键瓶颈是体内多种菌株的比较测试。我们将使用 由Co-I开发的新开发的高通量RNA和DNA“条形码”测序方法 该方法允许同时定量高达数百个转基因的转导效率和转基因表达， 在单个实验中使用AAV菌株来定义用于DRG神经元靶向的最佳载体特征。 目标2.开发磁共振成像（MRI）引导的靶向猪背根神经节的载体。 猪脊柱的MRI成像显示背根神经节的分界上级CT。磁共振成像- 可以在体模模型中校正所使用的针的错误定位伪影。我们将开发MRI引导的 AAV通过对流靶向DRG增强递送，同时将毒理学终点整合到目标中。 目标3.测试AAV在猪慢性膝骨关节炎（OA）疼痛模型中的镇痛功效。 膝关节OA是人类慢性疼痛的两个最常见原因之一。我们建立了一个 猪的慢性膝关节（“后膝关节”）OA。我们将使用该模型来测试镇痛效果和综合毒性 在DRG中表达阿片样物质基因的AAV的终点。 影响：沿着评估大型动物基因转移优化和有效性试验 毒理学终点将推进DRG的靶向镇痛AAV基因治疗的概念， 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。