CaMKII

慢病毒是以HIV-1为基础改造而来的一种病毒载体。在数十年的应用中，慢病毒载体的生物安全性和有效性不断被提升，目前已经成为体内外生物实验中一种功能非常强大的基因递送工具。 在基因递送方面，慢病毒载体拥有诸多独特的优势：稳定持久表达、可感染多种类型细胞、可携带大片段基因、免疫原性比较低等。无论是基础科研还是临床应用，慢病毒载体都具有非常广泛的应用前景。 慢病毒的应用场景 目前慢病毒载体主要应用在以下几个方面： 细胞基因治疗（截止目前，已上市的CAR-T产品有9款采用了慢病毒载体递送CAR基因） 基因表达调控（过表达、RNA干扰研究等） 基因编辑（CRlSPR/Cas9 gRNA文库筛选、基因敲除、敲入、点突变、基因激活/抑制） 稳转细胞株构建 活体细胞成像追踪 转基因动物 图1 慢病毒载体的主要应用方向 应用案例 案例1：用慢病毒载体进行基因过表达，构建稳转细胞株，研究葡萄糖激酶在前列腺癌（PCa）中的作用1 细胞代谢在肿瘤进展中起着关键作用，靶向癌症代谢可能有效地杀死癌细胞。本文旨在研究葡萄糖激酶在前列腺癌中的作用，并确定PCa治疗的关键靶点。 实验设计和主要研究结果： 癌症基因组图谱（TCGA）数据库和在线工具评估结果显示ADP依赖型葡萄糖激酶（ADPGK）是唯一一种在前列腺腺癌（PRAD）中上调并预测较差总生存（OS）的葡萄糖激酶。临床样本分析显示，与非PCa组织相比，ADPGK在PCa组织中显著上调。 ADPGK过表达促进了PCa细胞的增殖和迁移，而敲低ADPGK则抑制了恶性表型。 代谢组学、蛋白质组学以及ECAR和OCR测试揭示了ADPGK显著加速了PCa的糖酵解。 ADPGK与丙酮酸激酶C（ALDOC）结合，通过AMP激活蛋白激酶（AMPK）磷酸化促进糖酵解。 图2 用于ADPGK过表达的慢病毒结构示意图及ADPGK过表达结果。 案例2：用慢病毒载体递送RNA干扰工具，研究脯氨酸羟化酶结构域蛋白在调节心肌细胞钙水平中的功能2 由于高氧消耗，心肌细胞对环境中的氧气极为敏感。脯氨酸羟化酶结构域蛋白2（PHD2）是一种氧气传感器，对细胞缺氧适应性至关重要。近年来的研究发现，PHD抑制剂（PHIs）和针对PHD2的小干扰RNA（PHD2shRNA）可以在不影响细胞能量水平的情况下激活AMPK，将细胞缺氧信号转化为能量代谢信号，使细胞产生能量保护反应，并显著提高细胞对缺氧的抵抗力。研究还发现，信号分子钙（Ca2+）在响应缺氧的AMPK信号通路激活中发挥着重要作用。然而，PHD活性如何增加细胞内Ca2+水平并激活AMPK信号通路的分子机制仍然不清楚。 实验设计和主要研究结果： 确认PHIs 诱导心肌细胞中的钙释放。 PHD2 在心肌细胞中激活肌浆网通道辅助蛋白 CaMKⅡ。用shRNA干扰技术敲低心肌细胞中PHD2，Western blot证实了PHD2的沉默。与对照细胞相比，沉默PHD2显著增加了CaMKⅡ的磷酸化。 Ca2+ 在心肌细胞中介导 PHI 刺激引起的 AMPK 激活。肌浆网通道抑制剂瑞安诺丁显著降低了PHI处理诱导的与Ca2+相关的荧光强度。慢病毒介导的RyR2特异性shRNA显著降低了RyR2在mRNA水平上的表达。此外， IP3R阻断剂2-APB预处理或慢病毒介导的IP3R特异性shRNA均未显著影响PHI处理诱导的P-AMPK水平。2-APB预处理对PHI诱导的Ca2+荧光也无显著影响。这些结果表明，PHI诱导的钙释放和AMPK激活主要由肌浆网通道而非IP3R通道调节。 CaMKⅡ介导PHI诱导的钙释放和AMPK激活。PKA不参与PHI/Ca2+/AMPK信号通路，而CaMKⅡ在PHI/Ca2+/AMPK信号通路中发挥着重要作用。 TRPA1介导PHI/Ca2+/AMPK信号通路的激活。TRPA1抑制剂HC030031预处理显著抑制了PHI诱导的P-AMPK和P-CaMKⅡ增加，并显著减少了PHI诱导的Ca2+荧光。PHD2 shRNA组中TRPA1蛋白水平显著高于对照组。这些结果强烈表明TRPA1在PHD诱导的Ca2+释放途径中发挥着至关重要的作用。 图3 慢病毒介导的特异性shRNA对 PHD2、RyR2和IP3R的敲低效果。 案例3：用慢病毒介导的基因编辑技术研究穿透内耳血迷路屏障（BLB）的靶点3 内耳疾病是一种导致全球超过 15 亿人听力丧失的疾病。然而，内耳毛细血管表面存在血迷路屏障（BLB），由于渗透性低，限制了大多数药物化合物从血流进入内耳组织，极大地阻碍了全身药物预防和干预的有效性。本研究报告了一种在 BLB 上表达的新型受体低密度脂蛋白受体相关蛋白 1 (LRP1)，它可以作为穿梭治疗药物跨越这一屏障的潜在靶标。 实验设计和主要研究结果： LRP1在小鼠和猪的耳蜗细胞中广泛表达，尤其在BLB区域和目标细胞中。 通过靶向LRP1的配体肽IETP2，可以将不同的小分子如荧光染料、抗氧化药物和MRI造影剂等传递到内耳。这表明LRP1可能是一个有效的靶点，用于开发非侵入性和针对性的药物递送策略，以提高内耳疾病的治疗效果。 LRP1在细胞内吞作用中也起着关键作用。研究表明，IETP2肽与LRP1结合后可以被LRP1阳性细胞内化，并且这种内吞作用依赖于LRP1的表达。通过使用CRISPR/Cas9技术敲低LRP1基因，进一步证实了LRP1在IETP2内吞中的关键作用。 这些发现为开发新的治疗策略提供了重要的基础，可能有助于未来内耳疾病的诊断和治疗。通过利用LRP1的介导作用，研究人员可以探索将治疗药物更有效地传递到内耳，从而提高治疗效果并减少副作用。 图4 用于敲低LRP1的慢病毒载体结构和sgRNA。 图5 LRP1的敲低效果。 派真生物有幸为以上三项研究提供慢病毒。 案例4：慢病毒递送高灵敏度的报告基因Akaluc用于追踪胶质瘤的体内生长、侵袭和转移过程4 使用非侵入性生物发光成像（BLI）对肿瘤生长进行纵向追踪是研究体内癌症模型的关键方法。Akaluciferase（Akaluc）是一种新的BLI系统，其信号强度高于标准的萤火虫荧光素酶（Fluc）。本文设计了一种慢病毒载体，用于在胶质瘤细胞系中表达Akaluc，追踪胶质瘤的体内扩张过程。 实验设计和主要研究结果： Akaluc和Fluc报告基因的设计和灵敏度比较。用表达Venus-Akaluc或Venus-Fluc融合蛋白的慢病毒载体转导人GBM细胞系U87MG和小鼠高级别胶质瘤细胞系GL261，结果显示病毒载体转导对胶质瘤细胞的增殖率没有显著影响，表达Akaluc的细胞比表达Fluc的细胞的BLI信号强大约10倍。  颅内移植表达Akaluc的胶质瘤细胞产生超过Fluc 100倍的BLI信号。将GL261-Venus-Akaluc和GL261-MSCV-Fluc胶质瘤细胞颅内植入到同系的C57BL/6小鼠中，在所有时间点，Venus-Akaluc组的BLI信号显著高于GL261-Fluc组。 Akaluc BLI系统可以追踪更少数量的移植胶质瘤细胞，从而有助于更准确地模拟肿瘤发生和早期肿瘤生长。 Akaluc BLI系统可用于在体内对胶质瘤治疗反应进行纵向追踪。 总之，Akaluc BLI提供了一种敏感的方法用于体内追踪胶质瘤生长、侵袭和转移，特别适用于对较小的肿瘤进行成像以及监测治疗后的肿瘤复发。 图6 用于递送Akaluc BLI系统的慢病毒载体结构示意图以及Akaluc BLI系统体内示踪效果。 派真生物可提供表达Akaluc的慢病毒。 参考资料： https://doi-org.libproxy1.nus.edu.sg/10.1186/s40779-023-00500-9 https://doi-org.libproxy1.nus.edu.sg/10.1016/j.yexcr.2021.112777 https://www-nature-com.libproxy1.nus.edu.sg/articles/s41392-022-00995-z https://doi-org.libproxy1.nus.edu.sg/10.1007/s00259-021-05439-4 关于派真生物

‘Undruggable’ targets aren’t just in oncology. Cardurion Pharmaceuticals is hoping to become the first to successfully develop a treatment for a genetic heart condition that researchers have been studying for decades. The startup is working on a rare heart disease known as catecholaminergic polymorphic ventricular tachycardia — CPVT for short — that can lead to abnormal heart rhythms and is estimated to impact one in 10,000 people. To do so, it’s using a CaMKII inhibitor and has become the first to put such a drug into human studies. It’s not an easy target. CaMKII is a protein kinase that is involved in both heart function and human memory — meaning that any drug going after it has to be extra precise. Cardurion CEO Peter Lawrence told Endpoints News that big pharma companies they are in contact with “were really intrigued by the fact we were able to get into human clinical testing with the CaMKII inhibitor because other major pharmas and biotechs have tried for a very long time — and it was hard to do.” Cardurion’s plan to develop the next generation of heart disease treatments involves a mix of internal research and external hunting. Founded by cardiologist and seasoned industry executive Michael Mendelsohn, who previously led cardiovascular research at Merck Research Laboratories, Cardurion launched in 2017 through a partnership deal with Takeda. As part of that partnership, a cardiovascular research team from Takeda’s site in Shonan, Japan turned into Cardurion’s drug discovery unit. Along with its CaMKII inhibitor, the Massachusetts-based company has clinical trials for heart failure studying two PDE9 inhibitors, one of which was a failed blood disorder treatment that Cardurion is looking to revive. In May, the company touted biomarker results from its Phase 2 study of one of those inhibitors. The company declined to disclose exactly how much money it has raised, beyond saying it’s somewhere north of $500 million. That includes a $260 million Series B that Cardurion announced in July and part of a $300 million commitment from Bain. “What we do is translate our expertise in cardiovascular signaling pathways into therapeutics for our patients. That is the scientific thesis that undergirds everything that we do,” Lawrence said. “We are constantly thinking about what could be attractive targets to pursue in various diseases. And we don’t talk a lot about what we’re looking at, because it’s proprietary, but it is no different than oncology.” Key backers: Ascenta Capital, Blue Owl Capital, Delos Capital, Digitalis Ventures, Farallon Capital Management, Fidelity Management, GV, Millennium Management, New Enterprise Associates, The Invus Group, Bain Capital Life Sciences and Bain Capital Private Equity, Polaris Partners, Takeda Pharmaceuticals Find the full list of 2024 Endpoints 11 winners here .

Cardurion also plans to use the funding to expand the indications outlined for the two candidates and acquire additional assets for treating cardiovascular disorders. Image Credit: VectorMine / Shutterstock. US-based cardiology biotech Cardurion Pharmaceuticals has raised $260m in Series B financing to advance its drug pipeline. The proceeds will fund the late-stage trials for two of the company’s lead candidates, CRD-750, a phosphodiesterase 9 (PDE9) inhibitor for heart failure; and CRD-4730, a calmodulin-dependent protein kinase II (CaMKII) inhibitor for treating catecholaminergic polymorphic ventricular tachycardia. Both therapies are being evaluated in Phase II trials. The company also plans to use the fund to expand the number of indications being explored for the two candidates and acquire additional assets for treating cardiovascular disorders. The investors in the Series B round included Bain Capital, Ascenta Capital, and other US-based investment companies. CRD-750 is being evaluated in two Phase II trials (NCT06215586 and NCT06215911) to treat two types of heart failure – heart failure with preserved ejection fraction (HFpEF) and reduced ejection fraction (HFrEF). Both placebo-controlled trials are currently recruiting patients across multiple locations in the US and Canada. In May, the company presented positive data for another PDE9 inhibitor, CRD-740, in patients with heart failure. The CARDINAL-HF Phase IIa trial (NCT05409183) met its primary endpoint in patients with HFrEF by demonstrating a statistically significant median increase in plasma cyclic guanosine monophosphate (cGMP). See Also: Sumitomo Pharma’s DSP-5336 gains FDA fast track for AML AstraZeneca completes Amolyt Pharma acquisition CRD-4730 is being evaluated in a Phase II trial (NCT06005428) as a treatment for catecholaminergic polymorphic ventricular tachycardia—a rare genetic arrhythmic disease. The company also plans to develop CaMKII inhibitors to target additional cardiovascular indications. There has been an increased interest in developing novel cardiovascular therapies in recent months. In February, Novo Nordisk agreed to pay up to $1.46bn to license multiple molecular glue degraders for treating cardiometabolic disorders and rare diseases from Neomorph. As part of the agreement, Neomorph would bear discovery and preclinical research responsibilities while Novo Nordisk would have exclusive rights to further clinical development and commercialisation. Another approach that has emerged as a promising treatment in the cardiovascular space is drugs targeting the glucagon-like peptide-1 (GLP-1) receptor. Last year, Novo Nordisk reported that its GLP-1 therapy Wegovy (semaglutide) reduced the incidence of cardiovascular events in obese and overweight patients by 20% compared to placebo.