CD51

传统 LNP 包含 4 种成分:可电离脂质结合核酸分子，并协助其完成内体逃逸；两亲性磷脂促进细胞质膜和内体逃逸；胆固醇稳定 LNP 结构；PEG 脂质增强胶体稳定性并减少网状内皮细胞的清除。尽管传统的 LNP 安全有效，但是仅仅局限于肌内给药和针对肝细胞的静脉注射。传统 LNP 的局限性很大程度上源于其与极低密度脂蛋白的理化相似性以及吸附血浆中载脂蛋白 E 的倾向，导致其富集到肝脏中，并通过低密度脂蛋白受体被摄取到肝细胞中。传统 LNP 的这些特征虽说对肝脏类疾病的治疗是有益的，然而，严重阻碍了 LNP 在肝脏以外组织器官中的应用。 麻省理工学院 Langer 作为纳米递送领域化神级别的存在，多年以来，开枝散叶，门派蔚为可观，不少弟子已隐隐成为新一代宗主。Daniel Siegwart 也是一位从 Langer 和 Aderson 实验室走出来的悍将，他掌管的课题组位于美国德克萨斯大学西南医学中心 (UT Southwestern Medical Center)，长久以来一直专注于基因药物靶向性递送载体的开发，希望荡平递送障碍，发掘各种类型的 RNA（siRNA、miRNA、tRNA、mRNA）、质粒、基因编辑器等的体内治疗潜力，解决各种未被攻克的癌症、遗传性疾病、传染病等疾病。 随着基于 CRISPR 技术的基因编辑工具的爆发式迭代更新，人们寄希望于通过矫正基因组突变彻底攻克一系列人类遗传性疾病的困扰，例如镰状细胞病、血友病、囊性纤维化、杜氏肌营养不良症、遗传性失明，当然还有癌症。但是，CRISPR 系统有一个主要缺点：该系统由大分子组成，很难进入细胞。要想将 CRISPR 技术真正应用于临床治疗，必须首先找到高效精准的递送载体。 2016 年，麻省理工学院 Anderson 实验室 Hao Yin 等人采用脂质纳米颗粒递送 Cas9 mRNA，同时利用腺相关病毒编码 sgRNA 和修复模板，借助此种递送策略来治疗人类遗传性酪氨酸血症小鼠模型，并表明该治疗通过纠正致病性 Fah 剪接突变产生了延胡索酰乙酰乙酸水解酶 (Fah) 阳性肝细胞，延缓小鼠体重减轻和肝损伤等疾病症状。然而，这种递送策略的缺点是肝脏细胞的编辑效率非常低，只有 6%左右。 2016 年 12 月，Siegwart 成为第一个成功采用两性离子脂质纳米颗粒（ZNP）共递送 cas9 mRNA/sgRNA 进行基因编辑的研究者。2020 年， Siegwart 开发出一种称为选择性器官靶向纳米颗粒递送系统 SORT，可精确地将治疗分子递送至肝脏或者肝外组织中。SORT LNP 系统是在原有的 LNP 传统 4 组分脂质成分中加入了第 5 种脂质成分，从而实现 mRNA-LNP 特异性的组织和细胞靶向性。如果添加的是永久性的阳离子脂质，那么递送后的 mRNA 蛋白表达主要出现在肺部；如果添加的是永久性的阴离子脂质，将会促进 mRNA-LNP 更多地富集到脾脏中；添加可电离脂质则会增加向肝脏中的递送。也就说，通过调整 SORT 分子（第 5 种脂质分子）的化学性质和数量能够改变 LNP 的递送靶向性。凭借 SORT 组织靶向性纳米颗粒递送系统，Siegwart 成为首个完成体内组织特异性基因编辑的学者。 近日，Siegwart 在 Science 发表重磅文章：In vivo editing of lung stem cells for durable gene correction in mice，他利用 SORT LNP 系统递送碱基编辑器，可在囊肿性纤维化小鼠体内实现肺干细胞（50%比例）持久的基因矫正效果。最初，Siegwart 想采用靶向肺部的 SORT 纳米颗粒(由 5A2-SC8、胆固醇、DOPE、DMG-PEG 及 DOTAP 组成)将基因编辑器递送至肺部组织驻留干细胞中，重构拥有矫正 CFTR 突变基因的肺表皮细胞。因此，首先要采用报告基因确认肺部靶向性的 SORT 纳米颗粒确实可以将基因编辑系统递送至肺部细胞，并且发生正常的基因编辑功能。 在正常情况，Ail4 Loxp-stop-Loxp tdTomato（tdTom）报告基因小鼠上游存在的终止元件（loxP-Stop-loxP, LSL）会阻断 tdTomato 基因表达。Cre 重组酶或者 CRISPR-Cas9 会移除终止元件，激活 tdTom 报告基因的表达，从而用来表征发生基因编辑的细胞。间隔 48h，分两次向 Ail4 小鼠静脉注射 Cre mRNA-SORT LNP(2 mg/kg)，整个肺部组织的荧光信号表达从注射后第 2 天一直持续到第 660 天。在多种类型的肺部细胞中均可检测到持续的荧光信号，例如，内皮细胞，表皮细胞，免疫细胞，NGFR+阳性干细胞或者 KRT5+阳性干细胞。内皮细胞和肺部驻留免疫细胞的存活时间为几天至几周，但是，在这些细胞中经过基因编辑引发的荧光信号确可以持续至 1.8 年，这说明肺部靶向性 Cre mRNA-SORT LNP 使得肺部驻留内皮祖细胞和免疫祖细胞发生了基因编辑，最终产生了一大群经过编辑的成熟细胞。 此外，肺靶向性 SORT LNP 表面的蛋白冠层富含玻连蛋白（vitronectin），因此表达玻连蛋白受体（VtnR）的细胞摄取 SORT LNP 的效率会增强。与其他器官相比（VtnR 阳性率小于 4%），肺部是 VtnR 阳性率最高（24.4%）的器官，这也是肺靶向性 SORT LNP 递送特异性的一个重要原因。 囊肿性纤维化(Cystic Fibrosis, CF)是一种罕见的常染色体隐形遗传性疾病，主要影响肺部和消化系统。CFTR 基因突变是导致囊性纤维化的根本原因。CFTR 是一种 cAMP 依赖性的氯离子通道蛋白，在许多器官的各种上皮细胞中表达，包括肺、胰腺、肝脏、消化道和生殖道，可驱动氯离子向细胞外分泌。由于基因突变导致 CFTR 功能丧失，氯离子无法转运至细胞外，引起通道黏液异常黏稠，造成通道堵塞，肺气道内的阻塞引发感染。 研究人员用 ABE mRNA-LNP 转染源自 CF 病人的原代人支气管表皮细胞（HBE），此细胞携带 R553X-F508del 杂合突变。转染完成后，未分化的 P2 细胞培养物(主要由基底细胞构成)和 P3 细胞培养物会均会发生大约 60%的等位基因矫正。测序鉴定发现 T7 位置矫正为 C 的概率为 83.7%，邻近 T11 位置矫正为 C 的概率为 14.5%，T17 位置矫正为 C 的概率为 0。即便 T11 位置矫正为 C，也不会带来恶性后果，因为 GGT 和 GGC 均编码甘氨酸。 在携带 R553X 突变 CFTR 突变等位基因和 CFTR 正常等位基因的小鼠中，静脉注射一针 ABE mRNA LNP，肺干细胞基因组 T7 位置显示矫正概率为 50%，肺组织和气管组织中基因组 T7 位置矫正概率分别为 12.2%和 28.7%。 小结 利用基因 CRISPR 技术对肺部肝细胞进行基因编辑以实现遗传性肺部疾病的长久治疗效果，大家对此越来越感兴趣。Siegwart 课题组巧妙地在传统 LNP 配方中加入第 5 种额外的脂质，通过改变脂质纳米颗粒的理化特性，SORT LNP 模块化递送系统可成功实现肝外器官的靶向性递送。同时，结合 CRISPR 技术，初步对肺部遗传性疾病的实现了长久治疗效果。Siegwart 为将 SORT LNP 平台技术更好地应用于基因治疗药物的开发中，创立了 ReCode 公司，旨在开发具有精准靶向性的基因治疗药物。就体内基因编辑药物的研发来说，国内也处于非常领先的地位，尧唐生物在今年 3 月份宣布其自主研发的国内首个采用脂质纳米颗粒(LNP)递送的体内基因编辑药物 YOLT-201 的临床试验已获 CDE 批准。相信在未来 CRISPR 技术与靶向性 LNP 技术的强强联合一定会加速体内基因编辑药物的研发，攻克更多罕见的遗传性疾病。 识别微信二维码，添加生物制品圈小编，符合条件者即可加入 生物制品微信群！ 请注明：姓名+研究方向！ 版 权 声 明 本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

*仅供医学专业人士阅读参考 肺直接接触外部的“恶劣”环境，具备了超强的防御机制，可有效清除各种颗粒物和病原体。 但这也为经由气道的治疗带来了阻碍。理论上来说，由CFTR单基因失活突变导致的囊性纤维化（CF）非常适合基因治疗，但超过27项基因疗法都未能突破坚固的肺屏障而败走。 今日，《科学》杂志发表了来自德克萨斯大学西南医学中心科学团队的论文，研究者们这次“绕道而行”，选择从静脉给药，从基底膜一侧进入肺部，并在囊性纤维化小鼠中成功实现了体内编辑。 研究者使用了选择性肺靶向脂质纳米颗粒（lung SORT LNP），通过LNP组分优化避免了LNP在肝脏中的聚集。实验中，基因编辑成功矫正了小鼠肺干细胞中50%的CFTRR553X突变，有效增加了功能正常的CFTR水平。 论文题图 研究者首先测试了这种基因编辑方法的持久性，给小鼠静脉注射LNP，2mg/kg间隔48小时注射两次，随后定期收集肺组织进行分析。 lung SORT LNP的效果相当持久，在实验第660天仍旧能够观察到很高的编辑率。分析结果可见，第二次注射后，已经有超过32%的肺细胞被编辑，在第7天上升到51%，并持续至实验结束。 编辑效果可持续长达22个月 基因编辑也基本覆盖了小鼠肺部的主要细胞，包括内皮细胞、上皮细胞、免疫细胞和干细胞在内的多种细胞类型。 660天时，内皮细胞编辑率＞93%，免疫细胞＞23%、上皮细胞＞48%。 而在干细胞中，以NGFR为标志物，编辑率在实验第2天达到28%，180天时升高至75%，660天时仍能维持在45%到70%；以KRT5为标志物，编辑率在第2天达到27%，120天达到峰值97%，660天时维持在45%到80%。 在干细胞中能够维持不错的编辑效率 lung SORT LNP的安全性也很不错，在240天时检测，小鼠肾和肝功能正常，心脏、脾脏未见组织损伤。 研究者分析LNP转运机制发现，玻连蛋白会在LNP外形成类似斗篷的结构，使其更容易被有相应受体VtnR的细胞吸收，这也避免了LNP被免疫系统清除，有效地提高了编辑效率。 肺部各种细胞类型中VtnR表达比例都不低 接下来，研究者尝试了lung SORT LNP对囊性纤维化的治疗效果，目标是通过ABE将目前无法治疗的CFTR无义突变CFTRR553X编辑为野生型CFTR。 在人类囊性纤维化患者支气管上皮细胞中，LNP-ABE实现了约60%的编辑效率，成熟CFTR表达增加了5.5倍。将LNP-ABE与F508del突变患者的标准疗法联合使用，可将CFTR表达进一步增加至7.8倍。 基因编辑联合药物效果更佳 在人源化CFTRR553X突变小鼠中，1.5mg/kg的LNP-ABE静脉注射治疗10天后，可见肺干细胞中编辑率约50%，肺细胞总编辑率12.2%。 在小鼠中完成了体内编辑 这一比例看似不高，但已经足以带来有希望的临床增益。此前有研究结果表明，10-20%的CFTR表达细胞已经足以维持机体所需CFTR功能，相关临床试验设计目标为将CFTR功能提升至野生型的10%；就基因编辑来说，有研究证实，肺干细胞编辑率达到1.5%就足以起到治疗效果。 不过，小鼠并不具有相应的囊性纤维化表型，在更好的动物模型中进行实验，以确定疗效和安全性是必须的。 参考资料： [1]https://www-science-org.libproxy1.nus.edu.sg/doi/10.1126/science.adk9428 [2]https://www-science-org.libproxy1.nus.edu.sg/doi/10.1126/science.adq0059 本文作者丨代丝雨

Phase 1b/2a open-label trial in the U.S. of certepetide in combination with neoadjuvant FOLFIRINOX based therapies in pancreatic, colon and appendiceal cancers BASKING RIDGE, N.J., June 13, 2024 (GLOBE NEWSWIRE) -- Lisata Therapeutics, Inc. (Nasdaq: LSTA) (“Lisata” or the “Company”), a clinical-stage pharmaceutical company developing innovative therapies for the treatment of advanced solid tumors and other serious diseases, announced today the successful completion of patient enrollment for the pancreatic cancer cohort in the Phase 1b/2a CENDIFOX trial. This investigator-initiated trial, led by Dr. Anup Kasi at The University of Kansas (“KU”) Cancer Center, is evaluating the safety and efficacy of certepetide (formerly LSTA1) in combination with FOLFIRINOX-based therapies for pancreatic, colon, and appendiceal cancers. “The successful enrollment of the pancreatic cancer cohort represents another significant step forward in the clinical development of certepetide,” said Dr. Kristen K. Buck, Lisata’s Executive Vice President of Research and Development and Chief Medical Officer. “We are encouraged by the progress of the CENDIFOX trial and look forward to both the completion of enrollment in the remaining two cohorts and the reporting of results.” The open-label CENDIFOX trial is designed to assess the safety and therapeutic effects of combining certepetide with neoadjuvant FOLFIRINOX regimens, with or without panitumumab, across pancreatic, colon, and appendiceal cancers. The study, conducted solely at the KU Cancer Center, aims to enroll a total of 51 patients (21 resectable and borderline resectable pancreatic cancer patients, 15 high-grade colon and appendiceal cancer patients with peritoneal metastasis, and 15 colon cancer patients with oligo-metastatic disease). It will provide Lisata with valuable pre- and post-treatment tumor tissue data for immune profiling, along with long-term patient outcome information. The trial is funded by the KU Cancer Center and Lisata is supplying certepetide. “We are excited to collaborate with Lisata and encouraged by certepetide’s potentially diverse applicability,” said Dr. Anup Kasi, the study’s Principal Investigator at the KU Cancer Center. “We are eager to finish enrolling participants in the remaining study cohorts and to analyze the findings of each group to determine its efficacy.” For more information on the CENDIFOX trial, please visit https://www.clinicaltrials.gov/study/NCT05121038. About Certepetide Certepetide (formerly LSTA1) is an investigational drug designed to activate a novel uptake pathway that allows co-administered or tethered anti-cancer drugs to penetrate solid tumors more effectively. Certepetide actuates this active transport system in a tumor-specific manner, resulting in systemically co-administered anti-cancer drugs more efficiently penetrating and accumulating in the tumor. Certepetide also has the potential to modify the tumor microenvironment resulting in tumors which are more susceptible to immunotherapies. We and our collaborators have amassed significant non-clinical data demonstrating enhanced delivery of a range of emerging anti-cancer therapies, including immunotherapies and RNA-based therapeutics. To date, certepetide has also demonstrated favorable safety, tolerability, and clinical activity in completed and ongoing clinical trials designed to test its ability to enhance the effectiveness of standard-of-care chemotherapy for pancreatic cancer. Lisata is exploring the potential of certepetide to enable a variety of treatment modalities to treat a range of solid tumors more effectively. Certepetide has been awarded Fast Track designation (U.S.) and Orphan Drug Designation for pancreatic cancer (U.S. and E.U.) as well as Orphan Drug Designation for glioma (U.S.) and osteosarcoma (U.S.). Additionally, certepetide has received Rare Pediatric Disease Designation for osteosarcoma (U.S.). About Lisata Therapeutics Lisata Therapeutics is a clinical-stage pharmaceutical company dedicated to the discovery, development and commercialization of innovative therapies for the treatment of advanced solid tumors and other major diseases. Lisata’s lead product candidate, certepetide (formerly LSTA1), is an investigational drug designed to activate a novel uptake pathway that allows co-administered or tethered anti-cancer drugs to selectively target and penetrate solid tumors more effectively. Lisata has already established noteworthy commercial and R&D partnerships based on its CendR Platform® technology. The Company expects to announce numerous milestones over the next two years and believes that its projected capital will fund operations into early 2026, encompassing anticipated data milestones from its ongoing and planned clinical trials. For more information on the Company, please visit www.lisata.com. Forward-Looking Statements This communication contains “forward-looking statements” that involve substantial risks and uncertainties for purposes of the safe harbor provided by the Private Securities Litigation Reform Act of 1995. All statements, other than statements of historical facts, included in this communication regarding strategy, future operations, future financial position, future revenue, projected expenses and capital, prospects, plans, and objectives of management are forward-looking statements. In addition, when or if used in this communication, the words “may,” “could,” “should,” “anticipate,” “believe,” “estimate,” “expect,” “intend,” “plan,” “predict” and similar expressions and their variants, as they relate to Lisata or its management, may identify forward-looking statements. Examples of forward-looking statements include, but are not limited to, the potential efficacy of certepetide as a treatment for patients with metastatic pancreatic ductal adenocarcinoma and other solid tumors, statements relating to Lisata’s continued listing on the Nasdaq Capital Market; expectations regarding the capitalization, resources and ownership structure of Lisata; the approach Lisata is taking to discover and develop novel therapeutics; the adequacy of Lisata’s capital to support its future operations and its ability to successfully initiate and complete clinical trials; and the difficulty in predicting the time and cost of development of Lisata’s product candidates. Actual results could differ materially from those contained in any forward-looking statement as a result of various factors, including, without limitation: results observed from a single patient case study are not necessarily indicative of final results and one or more of the clinical outcomes may materially change following more comprehensive reviews of the data and as more patient data becomes available, including the risk that unconfirmed responses may not ultimately result in confirmed responses to treatment after follow-up evaluations; the risk that product candidates that appeared promising in early research and clinical trials do not demonstrate safety and/or efficacy in larger-scale or later clinical trials; the safety and efficacy of Lisata’s product candidates, decisions of regulatory authorities and the timing thereof, the duration and impact of regulatory delays in Lisata’s clinical programs, Lisata’s ability to finance its operations, the likelihood and timing of the receipt of future milestone and licensing fees, the future success of Lisata’s scientific studies, Lisata’s ability to successfully develop and commercialize drug candidates, the timing for starting and completing clinical trials, rapid technological change in Lisata’s markets, the ability of Lisata to protect its intellectual property rights; and legislative, regulatory, political and economic developments. The foregoing review of important factors that could cause actual events to differ from expectations should not be construed as exhaustive and should be read in conjunction with statements that are included herein and elsewhere, including the risk factors included in Lisata’s Annual Report on Form 10-K filed with the SEC on February 29, 2024, and in other documents filed by Lisata with the Securities and Exchange Commission. Except as required by applicable law, Lisata undertakes no obligation to revise or update any forward-looking statement, or to make any other forward-looking statements, whether as a result of new information, future events or otherwise. Contact: Investors: Lisata Therapeutics, Inc.John MendittoVice President, Investor Relations and Corporate CommunicationsPhone: 908-842-0084Email: jmenditto@lisata.com Media: ICR WestwickeElizabeth ColemanSenior AssociatePhone: 203-682-4783Email: elizabeth.coleman@westwicke.com