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AAV-delivered hepato-adrenal cooperativity in steroidogenesis: implications for gene therapy for congenital adrenal hyperplasia 类固醇生成过程中的肝-肾协同作用:先天性肾上腺皮质增生症基因疗法的意义
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-12 DOI: 10.1016/j.omtm.2024.101232
Lara E. Graves, Eva B. van Dijk, Erhua Zhu, Sundar Koyyalamudi, Tiffany Wotton, Dinah Sung, Shubha Srinivasan, Samantha L. Ginn, Ian E. Alexander
Despite the availability of life-saving corticosteroids for 70 years, treatment for adrenal insufficiency is not able to recapitulate physiological diurnal cortisol secretion and results in numerous complications. Gene therapy is an attractive possibility for monogenic adrenocortical disorders such as congenital adrenal hyperplasia, however, requires further development of gene transfer/editing technologies and knowledge of the target progenitor cell populations. Vectors based on adeno-associated virus are the leading system for direct gene delivery but have limitations in targeting replicating cell populations such as in the adrenal cortex. One strategy to overcome this technological limitation is to deliver the relevant adrenocortical gene to a currently targetable organ outside of the adrenal cortex. To explore this possibility, we developed a vector encoding human 21-hydroxylase and directed expression to the liver in a mouse model of congenital adrenal hyperplasia. This extra-adrenal expression resulted in reconstitution of the steroidogenic pathway. Aldosterone and renin levels normalised, and corticosterone levels improved sufficiently to reduce adrenal hyperplasia. This strategy could provide an alternative treatment option for monogenic adrenal disorders, particularly for mineralocorticoid defects. These findings also demonstrate, when targeting the adrenal gland, that inadvertent liver transduction should be precluded as it may confound data interpretation.
尽管拯救生命的皮质类固醇已问世 70 年,但肾上腺功能不全的治疗无法再现皮质醇的昼夜生理分泌,并导致许多并发症。基因疗法是治疗单基因肾上腺皮质疾病(如先天性肾上腺皮质增生症)的一种有吸引力的方法,但需要进一步开发基因转移/编辑技术,并了解目标祖细胞群。基于腺相关病毒的载体是直接传递基因的主要系统,但在靶向复制细胞群(如肾上腺皮质)方面存在局限性。克服这一技术限制的策略之一是将相关肾上腺皮质基因传递到肾上腺皮质以外的可靶向器官。为了探索这种可能性,我们开发了一种编码人类 21- 羟化酶的载体,并在先天性肾上腺增生症小鼠模型中将其定向表达到肝脏。这种肾上腺外表达导致了类固醇生成途径的重建。醛固酮和肾素水平恢复正常,皮质酮水平得到改善,足以减少肾上腺增生。这种策略可为单基因肾上腺疾病,尤其是矿质皮质激素缺陷提供另一种治疗选择。这些研究结果还表明,在以肾上腺为靶点时,应避免无意中进行肝脏转导,因为这可能会混淆数据解读。
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引用次数: 0
Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection 通过低顺式三重转染技术生产高数量和高质量的重组腺相关病毒
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-12 DOI: 10.1016/j.omtm.2024.101230
Hao Liu, Yue Zhang, Mitchell Yip, Lingzhi Ren, Jialing Liang, Xiupeng Chen, Nan Liu, Ailing Du, Jiaming Wang, Hao Chang, Hyejin Oh, Chen Zhou, Ruxiao Xing, Mengyao Xu, Peiyi Guo, Dominic Gessler, Jun Xie, Phillip WL. Tai, Guangping Gao, Dan Wang
Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that reduces the transgene plasmid usage by 10- to 100-fold, and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency, and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., ITR-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and widely applicable to producing rAAV of high quantity, purity, and potency.
基于重组腺相关病毒(rAAV)的基因疗法正以前所未有的速度进入临床和商业阶段。HEK293 细胞的三重转染是目前最广泛使用的 rAAV 生产平台。在这里,我们开发了低顺式三重转染技术,可将转基因质粒用量减少 10 到 100 倍,并克服了与标准三重转染相关的几个主要限制。这种新方法可将产量抑制型转基因的包装提高 10 倍,并能生产出减少质粒骨架污染的 rAAV 批次,而这种污染在下游处理过程中是无法消除的。在小鼠体内进行测试并与标准三重转染产生的 rAAV 进行比较时,低顺式 rAAV 显示出相当或更高的效力,并减少了质粒骨架 DNA 和 RNA 在组织中的持久性。从机理上讲,低顺式三重转染依赖于转基因盒(即 ITR 侧翼载体 DNA)在 HEK293 细胞中生产阶段的广泛复制。这种经济有效的方法易于实施,可广泛用于生产高数量、高纯度和高效力的 rAAV。
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引用次数: 0
Novel AAV variants with improved tropism for human Schwann cells 对人类许旺细胞具有更好趋向性的新型 AAV 变体
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-11 DOI: 10.1016/j.omtm.2024.101234
Matthieu Drouyer, Tak-Ho Chu, Elodie Labit, Florencia Haase, Renina Gale Navarro, Deborah Nazareth, Nicole Rosin, Jessica Merjane, Suzanne Scott, Marti Cabanes-Creus, Adrian Westhaus, Erhua Zhu, Rajiv Midha, Ian E. Alexander, Jeff Biernaskie, Samantha L. Ginn, Leszek Lisowski
Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of bespoke AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various , , and models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.
基因疗法和相关技术正在改变生物医学研究,并为衰弱和无法治愈的遗传疾病患者提供了新的治疗方案。以重组腺相关病毒载体(AAV)为基础的载体系统在最近的临床试验中显示出治疗肝脏和神经系统等多器官遗传疾病的巨大前景。尽管最近在开发新型生物工程 AAV 变体以改善原代人体组织和细胞的转导方面取得了成功,但能有效转导人许旺细胞(hSCs)的载体仍有待鉴定。在此,我们报告了在原代间充质干细胞中应用功能性转导-RNA选择方法开发定制AAV变体,以特异、高效地向间充质干细胞传递转基因。两种已鉴定的病毒壳变体--Pep2hSC1 和 Pep2hSC2--显示出在不同的造血干细胞模型中具有一致的转导效力。这些新型 AAV 胶囊将成为有价值的研究工具,为 SC 相关疾病或外周神经系统损伤的治疗方案奠定基础。
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引用次数: 0
Non-canonical capsid engineering highlights new possibilities for AAV vectorology 非典型囊壳工程彰显 AAV 载体学的新可能性
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-06 DOI: 10.1016/j.omtm.2024.101221
Zehan Zhang, John R. Counsell
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引用次数: 0
A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency 用于生产 AAV 向量的稳健而灵活的杆状病毒-昆虫细胞系统,可提高产量、囊壳比率和效力
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-04 DOI: 10.1016/j.omtm.2024.101228
Yoko Marwidi, Hoang-Oanh B. Nguyen, David Santos, Tenzin Wangzor, Sumita Bhardwaj, Gabriel Ernie, Gregg Prawdzik, Garrett Lew, David Shivak, Michael Trias, Jada Padilla, Hung Tran, Kathleen Meyer, Richard Surosky, Alex Michael Ward
Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system—the SGMO Helper—with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, and , compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.
用于基因和细胞治疗的腺相关病毒(AAV)的生产量大幅增加,促进了基于哺乳动物和昆虫细胞的改良生产系统的开发。我们开发了一种基于杆状病毒的昆虫细胞生产系统--SGMO Helper,它具有新颖的基因结构和更大的灵活性,可以调节单个 Rep 蛋白和 Cap 蛋白的表达水平和含量。此外,我们还对 AAV6 的囊膜序列进行了修改,从而提高了产量、囊膜完整性和效力。与科廷实验室的 Bac-RepCap 助手相比,使用 SGMO 助手生产重组 AAV6(rAAV6)的产量有所提高。与使用 Bac-RepCap 生产的 rAAV6 相比,SGMO Helper 衍生的 rAAV6 能抵抗先前描述的一种特有于杆状病毒-昆虫细胞生产系统的蛋白酶裂解,并且提高了囊膜比率和效力。下一代测序序列分析表明,SGMO Helper 经过六次连续传代后是稳定的,而且 rAAV6 荚膜中的非载体基因组 DNA 含量与使用 Bac-RepCap 生产的 rAAV6 相当。使用 SGMO Helper 生产 AAV 可通过生物反应器进行扩展,并能提高产量、囊壳比率和效力。我们的研究表明,SGMO Helper 是一种改进的 AAV 生产平台,可用于提供最先进的基因和细胞疗法。
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引用次数: 0
Embryo and fetal gene editing: Technical challenges and progress toward clinical applications 胚胎和胎儿基因编辑:技术挑战与临床应用进展
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-03-04 DOI: 10.1016/j.omtm.2024.101229
Citra N.Z. Mattar, Wei Leong Chew, Poh San Lai
Gene modification therapies (GMTs) are slowly but steadily making progress toward clinical application. As the majority of rare diseases have an identified genetic cause, and as rare diseases collectively affect 5% of the global population, it is increasingly important to devise gene correction strategies to address the root causes of the most devastating of these diseases and to provide access to these novel therapies to the most affected populations. The main barriers to providing greater access to GMTs continue to be the prohibitive cost of developing these novel drugs at clinically relevant doses, subtherapeutic effects, and toxicity related to the specific agents or high doses required. strategy and treating younger patients at an earlier course of their disease could lower these barriers. Although currently regarded as niche specialties, prenatal and preconception GMTs offer a robust solution to some of these barriers. Indeed, treating either the fetus or embryo benefits from economy of scale, targeting pre-pathological tissues in the fetus prior to full pathogenesis, or increasing the likelihood of complete tissue targeting by correcting pluripotent embryonic cells. Here, we review advances in embryo and fetal GMTs and discuss requirements for clinical application.
基因修饰疗法(GMT)正缓慢而稳步地走向临床应用。由于大多数罕见病都有已查明的遗传原因,而且罕见病总共影响全球 5% 的人口,因此制定基因矫正策略以解决最具破坏性的疾病的根本原因,并让受影响最严重的人群获得这些新型疗法就变得越来越重要。让更多人获得 GMTs 治疗的主要障碍仍然是开发临床相关剂量的新型药物的高昂成本、亚疗效以及与特定药物或所需大剂量有关的毒性。 在疾病的早期阶段对年轻患者进行治疗的策略可以降低这些障碍。尽管产前和孕前 GMT 目前被视为利基专科,但它为解决其中一些障碍提供了强有力的解决方案。事实上,对胎儿或胚胎进行治疗可从规模经济中获益,在完全发病之前靶向胎儿的病理前组织,或通过纠正多能胚胎细胞增加完全靶向组织的可能性。在此,我们回顾了胚胎和胎儿 GMTs 的进展,并讨论了临床应用的要求。
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引用次数: 0
Peptide-encoding gene transfer to modulate intracellular protein‒protein interactions 肽编码基因转移调节细胞内蛋白质与蛋白质之间的相互作用
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-02-28 DOI: 10.1016/j.omtm.2024.101226
Toshihiko Taya, Daisuke Kami, Fumiya Teruyama, Satoaki Matoba, Satoshi gojo
Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein‒protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.
肽类药物的发现具有巨大潜力,但当目标是细胞内蛋白质-蛋白质相互作用(PPI)时,细胞膜是一个主要障碍。小分子药物很难靶向 PPI;事实上,目前还没有针对任何细胞内 PPI 的干预工具。在这项研究中,我们开发了一种平台,可以通过基于 mRNA 的基因递送将肽引入细胞。肽长的核酸不能与核糖体稳定结合,几乎不能翻译成蛋白质。在这项研究中,我们创建了一种构建体,将编码二氢叶酸还原酶(DHFR)的序列放在编码目标多肽的序列前面,并加上一个翻译跳转序列,以满足促进核糖体结合和翻译蛋白质快速衰减的要求。这样就能从编码目标蛋白质的 mRNA 开始高效翻译,同时避免不必要的蛋白质残基。通过使用这种构建体,我们发现它可以抑制 Drp1/Fis1 的结合,而 Drp1/Fis1 是细胞内的 PPIs 之一,它控制着线粒体的裂变,这是线粒体动力学的一个重要方面。此外,它还能抑制病理性过度裂变,使线粒体动力学和新陈代谢正常化,并抑制线粒体途径的细胞凋亡。
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引用次数: 0
Manufacturing DNA in E. coli yields higher fidelity DNA than in vitro enzymatic synthesis 与体外酶法合成相比,在大肠杆菌中制造 DNA 可获得保真度更高的 DNA
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-02-28 DOI: 10.1016/j.omtm.2024.101227
Steven J. Hersch, Siddarth Chandrasekaran, Jamie Lam, Nafiseh Nafissi, Roderick A. Slavcev
Biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceuticals. The quality of starting material plays a pivotal role in determining final product quality. Here we examined the fidelity of DNA replication using enzymatic methods () compared to plasmid DNA produced in . Next-generation sequencing approaches rely on polymerases, which have inherent limitations in sensitivity. To address this challenge, we introduce a novel assay based on loss-of-function (LOF) mutations in the conditionally toxic gene. Our findings show that DNA production in results in significantly fewer LOF mutations (80- to 3000-fold less) compared to enzymatic DNA replication methods such as PCR and rolling circle amplification (RCA). These results suggest that using DNA produced by PCR or RCA may introduce a substantial number of mutation impurities, potentially affecting the quality and yield of final pharmaceutical products. Our study underscores that DNA synthesized has a significantly higher mutation rate than DNA produced traditionally in . Therefore, utilizing enzymatically-produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while using DNA starting material derived from substantially mitigates this risk.
基因治疗等生物技术将 DNA 载体推向了制药业的前沿。起始材料的质量在决定最终产品质量方面起着至关重要的作用。在这里,我们研究了使用酶法()复制 DNA 的保真度,并与......中生产的质粒 DNA 进行了比较。下一代测序方法依赖于聚合酶,而聚合酶在灵敏度方面存在固有的局限性。为了应对这一挑战,我们引入了一种基于条件毒性基因功能缺失(LOF)突变的新型检测方法。我们的研究结果表明,与聚合酶链式反应(PCR)和滚动圈扩增(RCA)等酶DNA复制方法相比,DNA生产过程中产生的LOF突变明显较少(少80-3000倍)。这些结果表明,使用 PCR 或 RCA 生产的 DNA 可能会引入大量突变杂质,从而可能影响最终药品的质量和产量。我们的研究强调,合成 DNA 的突变率明显高于传统方法生产的 DNA。因此,在生物技术和生物制造中使用酶法生产的 DNA 可能会带来相当大的与保真度相关的风险,而使用来自的 DNA 起始材料则大大降低了这种风险。
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引用次数: 0
Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart 通过血管内移植 mRNA 增强间充质基质细胞,猪心脏的治疗性蛋白表达效果更佳
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-02-27 DOI: 10.1016/j.omtm.2024.101225
Jonathan Al-Saadi, Mathias Waldén, Mikael Sandell, Jesper Solmér, Rikard Grankvist, Ida Friberger, Agneta Andersson, Mattias Carlsten, Kenneth Chien, Johan Lundberg, Nevin Witman, Staffan Holmin
Heart failure has a poor prognosis and no curative treatment exists. Clinical trials are investigating gene- and cell-based therapies to improve cardiac function. The safe and efficient delivery of these therapies to solid organs is challenging. Herein, we demonstrate the feasibility of using an endovascular intramyocardial delivery approach to safely administer mRNA drug products and perform cell transplantation procedures in swine. Using a -vessel wall (TW) device, we delivered chemically modified mRNAs (modRNA) and mRNA-enhanced mesenchymal stromal cells expressing vascular endothelial growth factor A (VEGF-A) directly to the heart. We monitored and mapped the cellular distribution, protein expression, and safety tolerability of such an approach. The delivery of modRNA-enhanced cells via the TW device with different flow rates and cell concentrations marginally affect cell viability and protein expression . Implanted cells were found within the myocardium for at least 3 days following administration, without the use of immunomodulation and minimal impact on tissue integrity. Finally, we could increase the protein expression of VEGF-A over 500-fold in the heart using a cell-mediated modRNA delivery system compared with modRNA delivered in saline solution. Ultimately, this method paves the way for future research to pioneer new treatments for cardiac disease.
心力衰竭的预后很差,目前尚无根治的疗法。临床试验正在研究基于基因和细胞的疗法,以改善心脏功能。将这些疗法安全有效地输送到实体器官是一项挑战。在这里,我们展示了使用血管内心肌内给药方法在猪体内安全给药 mRNA 药物产品和进行细胞移植手术的可行性。我们使用血管壁(TW)装置将化学修饰的 mRNA(modRNA)和表达血管内皮生长因子 A(VEGF-A)的 mRNA 增强间充质基质细胞直接输送到心脏。我们对这种方法的细胞分布、蛋白表达和安全耐受性进行了监测和绘图。通过 TW 装置输送 modRNA 增强细胞,不同的流速和细胞浓度对细胞存活率和蛋白表达影响不大。植入的细胞在给药后至少 3 天内仍在心肌内,无需使用免疫调节,对组织完整性的影响极小。最后,与在生理盐水中递送 modRNA 相比,我们利用细胞介导的 modRNA 递送系统可使心脏中 VEGF-A 蛋白表达量增加 500 倍以上。最终,这种方法为未来研究开创心脏疾病的新疗法铺平了道路。
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引用次数: 0
Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy 自动制造用于急性髓细胞性白血病治疗的 ΔNPM1 TCR 工程 T 细胞
IF 4.7 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Pub Date : 2024-02-27 DOI: 10.1016/j.omtm.2024.101224
Isabella Elias Yonezawa Ogusuku, Vera Herbel, Simon Lennartz, Caroline Brandes, Eva Argiro, Caroline Fabian, Carola Hauck, Conny Hoogstraten, Sabrina Veld, Lois Hageman, Karin Teppert, Georgia Koutsoumpli, Marieke Griffioen, Nadine Mockel-Tenbrinck, Thomas Schaser, Rosa de Groot, Ian C.D. Johnston, Dominik Lock
Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1-TCR CD8 T cells manufactured with the optimized process showed specific killing of AML and . The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.
急性髓性白血病(AML)是一种异质性恶性肿瘤,需要进一步改进治疗方法,尤其是针对老年人和预后不良的亚群。最近发现的一种靶向突变型核嗜磷蛋白 1(ΔNPM1)的 T 细胞受体(TCR)为开发癌症抗原靶向细胞疗法提供了一种极具吸引力的选择。然而,TCR修饰T细胞的制造仍然受到复杂、耗时和费力的程序的限制。因此,本研究特别针对ΔNPM1特异性T细胞的规模化生产要求,采用了自动化、封闭式和符合良好生产规范的流程。从低温保存的白细胞开始,对 2E8 CD8 阳性 T 细胞进行富集、激活、慢病毒转导、扩增,最后进行配制。通过调整和优化培养条件,我们还将制造时间从 12 天缩短到了 8 天,同时还获得了高达 5.5E9 ΔNPM1 TCR 工程 T 细胞的临床相关产量。细胞产品主要由具有早期记忆表型的高活性 CD8 阳性 T 细胞组成。用优化工艺制造的ΔNPM1-TCR CD8 T细胞对急性髓细胞白血病和白血病有特异性杀伤作用。该工艺已用于即将开展的治疗 NPM1 突变 AML 的 1/2 期临床试验。
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引用次数: 0
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Molecular Therapy-Methods & Clinical Development
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