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

腺相关病毒(AAV)基因疗法有可能提供靶向肛门--目前尚未实现-- 如果优化的载体可以在大型动物身上实现一个关键目标：高效，那么GERIC药物在疼痛患者中的传递 以及向背根神经节(DRG)神经元传递解剖学上选择性的基因。这样的基因打靶 甚至可以将阿片类药物(基因编码的)变成一种安全和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，每年花费5600亿美元。使用非靶向阿片类药物 治疗疼痛，如口服羟考酮作用于除背根神经节以外的多种神经结构，引起副作用 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，18893名美国人死于处方阿片类药物，190万人上瘾。 AAV基因治疗疼痛所需的关键技术已经存在，如多个治疗基因和 明确定义目标细胞群，即背根神经节神经元。事实上，DRG导向的AAV的镇痛效果 基因载体已经在啮齿动物模型中得到了彻底的验证。然而，令人惊讶的是，大型动物试验 没有关于任何止痛基因载体的报道，也没有AAV或任何其他载体类型的报道。 假设：解剖学上选择性地传递编码已知止痛基因的AAV载体以选择DRG 可以逆转临床上常见的局部区域疼痛综合征的大型动物模型的疼痛相关行为。 目的1.通过对猪背根神经节病毒株的体内高通量检测，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开发的高通量RNA和DNA条形码测序新方法 这使得同时量化转基因效率和多达数百个转基因表达 在单个实验中确定用于DRG神经元靶向的最佳载体特性。 目的2.建立磁共振成像(MRI)引导下的猪背根神经节靶向载体。 与CT相比，猪脊柱MRI图像显示背根神经节的分界更好。核磁共振- 使用的针的错误定位伪影可以在体模模型中得到纠正。我们将开发核磁共振引导的 AAV通过对流增强递送以DRG为靶点，同时将毒理学终点整合到目标中。 目的3.在猪膝关节慢性骨性关节炎(OA)疼痛模型上检测AAV的镇痛效果。 膝骨性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 猪慢性膝关节(“窒息”)骨性关节炎。我们将使用该模型来测试镇痛效果和综合毒性。 AAV在背根节表达阿片类基因的终点。 影响：执行大型动物基因转移优化和有效性测试，同时评估 毒理学终点将推进DRG靶向止痛性AAV基因治疗的概念 在那里可以启动IND前的讨论，并证明对正式毒理学测试的额外投资是合理的。

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.