A novel adult neurons screening technology to repurpose FDA-approved drugs for spinal cord injury

约有130万美国人因脊髓损伤(SCI)而瘫痪，给我们的经济造成了数十亿美元的损失 每年。到目前为止，还没有FDA批准的脊髓损伤治疗方案，这表明需要新的治疗方案。 减少神经细胞死亡和增强成年皮层神经元在不同部位形成连接的再生能力 脊髓通过皮质脊髓束的水平是促进脊髓损伤后恢复的关键。理想情况下，候选人治疗 应对皮质神经细胞(CNCs)进行筛查。然而，目前的筛查技术仅利用 胚胎或出生后早期的神经细胞，不代表脊髓损伤患者的人口统计，他们中的大多数要么 在20多岁或60多岁，分别相当于6个月和18个月大的小鼠。使用具有以下特征的神经细胞 与临床环境中的靶细胞不同，不考虑年龄，导致翻译成功率较低。 此外，目前的筛查不区分性别，而神经疾病和创伤可能与性别有关(78% 的脊髓损伤患者为男性)。与年龄和性别相适应的药物筛查以前并不可信，导致错误的 阳性和阴性可以解释许多药物在临床阶段失败的原因。开发一种屏幕摄像 年龄和性别作为变量会增加翻译成功的机会。 我们的长期目标是开发新的治疗方案，促进脊髓损伤患者的康复。主要目标 这项建议的目的是在临床前脊髓损伤小鼠模型中寻找新的化合物来提高恢复。三步曲 建立了以品种、年龄、性别为变量的成年绵羊和小鼠CNCS筛选平台。这是 第一个以高通量方式使用CNCS的屏幕，以及第一个能够使用大型哺乳动物脑组织的屏幕。 这项技术减少了处理时间和动物使用，同时在增加的神经元上提供了大量的CNCs 性价比高的筛查的纯度；2)找到对成年人有益的化合物，这些化合物被其他筛查过早排除；以及3) 决定了筛选出的化合物(老鼠和绵羊)的种间效力，增加了 对人类有效。在2岁成年绵羊CNCS中进行了1,200种独特化合物的靶向筛选 (在神经疾病的临床测试中识别药物)和6个月和18个月大的雄性/雌性小鼠的CNCS。4. 在脊髓损伤小鼠模型中检测到阳性命中，3例促进了功能恢复。这验证了这项技术的使用 寻找具有临床前疗效和潜在临床转译能力的化合物。 最重要的假设是，在不同物种、年龄和性别群体的成人中枢神经系统中筛选批准的药物 将通过以下方式提高临床前成功率：1)发现以前被废弃或未经测试的有益药物 常规筛查，以及2)识别与人口统计无关的疗效的药物。这项提案将筛选 L1000批准的药物资料库(&gt；2800种药物)，以寻找4个线索(目标1)并确定它们在促进功能方面的有效性 临床相关的脊髓损伤小鼠模型的康复(目标2)。在研究结束时，至少有一种新药可以增强 复苏将会被揭开。未来的研究将包括了解该药物的细胞和分子机制。 并收集支持IND的数据，用于未来在日益老龄化的脊髓损伤人群中进行临床测试。

About 1.3 million Americans suffer from paralysis due to spinal cord injury (SCI) costing our economy billions of dollars each year. To date, no FDA-approved therapeutic options exist for SCI, demonstrating the need for new therapeutic options. Reducing neuronal cell death and enhancing regenerative capacities of adult cortical neurons to form connections at different levels of the spinal cord via the corticospinal tract are keys to increasing recovery after SCI. Ideally, candidate therapeutic screenings should be conducted on cortical neural cells (CNCs). However, current screening technologies only utilize embryonic or early post-natal neural cells, which do not represent the SCI patient demographic, majority of whom are either in their 20s or 60s, equivalent to mice of 6 and 18 months of age, respectively. Using neural cells with characteristics that differ from the targeted cells in clinical settings, without taking age into consideration, results in low translational success. Additionally, current screens do not differentiate for sex, while neurological diseases and trauma can be sex dependent (78% of SCI patients are male). Age- and sex-appropriate drug screens have previously not been plausible resulting in false positives and negatives which can explain the failure of many drugs during the clinical phases. Developing a screen taking age and sex as variables would increase the chance of translational success. Our long-term goal is to develop novel therapeutic options that enhance recovery for patients with SCI. The main objective during this proposal is to find new compounds that improve recovery in a pre-clinical mouse model of SCI. A 3-step screening platform using adult sheep and mice CNCs that includes species, age and sex as variables was developed. This is the first screen using CNCs in a high-throughput fashion and the first capable of using brain tissue from large mammals. This technology 1) reduces processing time and animal use while providing a high number of CNCs at increased neuron purity for a cost-effective screen; 2) finds compounds beneficial to adults prematurely dismissed by other screens; and 3) determines the interspecies efficacy of the screened compounds mouse and sheep), increasing the likelihood of being effective in humans. A targeted screen of >1,200 unique compounds was conducted in 2-years-old adult sheep CNCs (identifying drugs in clinical testing for neurological disorders) and in 6 and 18-month-old male/female mice CNCs. 4 positive hits were tested in a mouse model of SCI, 3 promoted functional recovery. This validates the use of this technology to find compounds with pre-clinical efficacy and potential clinical translation. The overarching hypothesis is that screening of approved drugs in adult CNCs from various species, age and sex groups will increase the pre-clinical success rate by 1) uncovering beneficial drugs previously dismissed or untested in conventional screens, and 2) identifying drugs with demographics-independent efficacies. This proposal will screen the L1000 Approved Drug Library (>2,800 drugs) to find 4 leads (Aim 1) and determine their efficacy in promoting functional recovery in a clinically relevant mouse model of SCI (Aim 2). At the end of the studies, at least one novel drug enhancing recovery will be uncovered. Future studies will include understanding the cellular and molecular mechanisms of the drug and gathering IND-enabling data for future clinical testing in the ever-aging SCI population.