人类诱导多能干细胞衍生产品用于自体细胞治疗帕金森病的临床前安全性和有效性

•Clinical-grade hiPSCs were successfully generated from four PD patients

从 4 例 PD 患者中成功生成临床级 hiPSC

•These hiPSC-derived mDA cells showed no adverse effects in mice up to 9 months

这些 hiPSC 来源的 mDA 细胞在长达 9 个月的小鼠中没有显示出不良反应

•Grafts of mDA cells showed varied efficacy, highlighting individual differences

mDA 细胞移植物显示出不同的疗效，突出了个体差异

•An autologous phase 1 clinical trial is underway to treat 8 PD patients

一项治疗 8 名 PD 患者的自体 1 期临床试验正在进行中

人诱导多能干细胞 （hiPSC） 衍生的中脑多巴胺能细胞 （mDAC） 代表了帕金森病 （PD） 自体细胞治疗的有前途的来源，但标准化的监管标准对于临床转化至关重要。在这项临床前研究中，我们使用游离型重编程从四名散发性 PD 患者的新鲜活检成纤维细胞中生成了多个临床级 hiPSC 细胞系，并使用改进的 21 天方案将它们分化为 mDAC。严格的评估包括全基因组/外显子组测序、RNA 测序和体内研究，包括一项为期 39 周的符合良好实验室规范的小鼠安全性研究。虽然来自所有品系的 mDAC 都符合安全标准，但来自一名患者的 mDAC 未能改善啮齿动物的行为结果，强调了个体间的差异性。重要的是，体外评估并不能可靠地预测体内疗效，将多巴胺能纤维密度确定为关键疗效标准。这些发现支持了自体细胞疗法的综合质量控制指南，并为计划于 2025 年开始的 8 名散发性 PD 患者的临床试验铺平了道路。

Human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic cells (mDACs) represent a promising source for autologous cell therapy in Parkinson’s disease (PD), but standardized regulatory criteria are essential for clinical translation. In this pre-clinical study, we generated multiple clinical-grade hiPSC lines from freshly biopsied fibroblasts of four sporadic PD patients using episomal reprogramming and differentiated them into mDACs using a refined 21-day protocol. Rigorous evaluations included whole-genome/exome sequencing, RNA sequencing, and in vivo studies, including a 39-week Good Laboratory Practice-compliant mouse safety study. While mDACs from all lines met safety criteria, mDACs from one patient failed to improve rodent behavioral outcomes, underscoring inter-individual variability. Importantly, in vitro assessments did not reliably predict in vivo efficacy, identifying dopaminergic fiber density as a key efficacy criterion. These findings support comprehensive quality control guidelines for autologous cell therapy and pave the way for a clinical trial with eight sporadic PD patients, scheduled to commence in 2025.

Introduction   介绍

帕金森病 （PD） 是最普遍的神经退行性运动障碍，通常在晚年被诊断出来。截至 2016 年，全球有超过 610 万人受到影响，预计其发病率将随着人口老龄化而上升。尽管有许多旨在缓解其症状的治疗方式，但现有的干预措施并不能阻止或逆转疾病进展。因此，对新型治疗方法存在大量未满足的需求。

Parkinson’s disease (PD) is the most prevalent neurodegenerative movement disorder, typically diagnosed later in life. As of 2016, it affected over 6.1 million people worldwide, with its incidence projected to rise alongside the aging population.1,2 Despite numerous treatment modalities aimed at alleviating its symptoms, existing interventions do not arrest or reverse disease progression.3 Hence, there exists a substantial unmet need for novel therapeutic approaches.

PD 的一个标志是黑质内中脑多巴胺能神经元 （mDAN） 的进行性变性，导致多巴胺神经传递受损，并与 PD 的特征性运动缺陷直接相关。这一观察激发了 PD 细胞替代疗法 （CRT） 的概念，自 1980 年代以来对产生多巴胺的细胞移植方法进行了研究。   其中，人类胎儿脑组织移植在长时间缓解运动症状方面显示出前景，作为 CRT 的概念证明。然而，两项双盲、假对照研究揭示了可变且不显著的临床益处，以及偶尔的移植物诱导的运动障碍。这些早期研究强调了 CRT 使用更明确和受控的细胞产品的潜在优势，这可能为 PD 提供卓越的益处。因此，研究人员正在研究更实用的细胞来源，包括人胚胎干细胞 （hESC） 和人诱导多能干细胞 （hiPSC） 作为潜在的替代品。

A hallmark of PD is the progressive degeneration of midbrain dopaminergic neurons (mDANs) within the substantia nigra, resulting in impaired dopamine neurotransmission and directly correlating with PD’s characteristic motor deficits.4,5,6 This observation spurred the concept of cell replacement therapy (CRT) for PD, with investigations into dopamine-producing cell transplantation methods since the 1980s.7,8,9,10 Among these, human fetal brain tissue transplantation has shown promise in alleviating motor symptoms for extended periods, serving as a proof of concept for CRT.7,8,9,10,11,12,13,14 However, two double-blind, sham-controlled studies revealed variable and insignificant clinical benefits, alongside occasional graft-induced dyskinesia.15,16 These earlier studies underscored the potential advantages of CRT using more defined and controlled cell products, which may offer superior benefits for PD. Consequently, researchers are investigating more practical cell sources, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), as potential alternatives.

源自个体 PD 患者的 hiPSC 有望成为自体个性化 CRT 的细胞来源。 为了探索这种潜力，我们开发了一个全面的平台，涵盖体细胞重编程的分子机制、 参与代谢重编程的 microRNA 簇的鉴定以及重编程和体外分化方法的改进。 此外，我们还建立了消除未分化 hiPSC 的化学方法并改进了手术移植技术。 利用该平台，我们成功地从散发性 PD 患者“PD01”中生成了 hiPSC 系，并将其分化为中脑多巴胺能细胞 （mDAC），其中主要包括中脑多巴胺能祖细胞 （mDAP） 和早期 mDAN。经过严格的安全性和有效性评估， 我们获得了美国食品药品监督管理局 （FDA） 的批准，扩大了单患者治疗 PD01 的可及性。在这项初步研究中，我们在良好生产规范设施中生成了 mDAC，并将其移植到患者的壳核中，没有免疫抑制。使用氟-18-L-二羟基苯丙氨酸 （ 18 F-DOPA） 的正电子发射断层扫描显示移植物在 18-24 个月时存活。 患者无不良反应，症状有适度改善。

hiPSCs, derived from individual PD patients, hold promise as cell sources for autologous, personalized CRT.17,18 To explore this potential, we have developed a comprehensive platform spanning molecular mechanisms underlying somatic cell reprogramming,19,20 identification of microRNA clusters involved in metabolic reprogramming,21 and refining of reprogramming and in vitro differentiation methods.21,22 Additionally, we have established chemical methods to eliminate undifferentiated hiPSCs21,23 and improved surgical transplantation techniques.24 Utilizing this platform, we successfully generated hiPSC lines from a sporadic PD patient, “PD01,” and differentiated them into midbrain dopaminergic cells (mDACs),21,22 which predominantly comprise midbrain dopaminergic progenitors (mDAPs) and early mDANs. After rigorous safety and efficacy assessments,21 we obtained U.S. Food and Drug Administration (FDA) approval for single-patient expanded access to treat patient PD01. In this pilot study, we generated mDACs in a Good Manufacturing Practice facility and transplanted them into the patient’s putamen without immunosuppression. Positron emission tomography scans using fluorine-18-L-dihydroxyphenylalanine (18F-DOPA) indicated graft survival at 18–24 months.25 The patient experienced no adverse effects and modest symptom improvement.

基于本病例和 FDA 建议，我们提出了临床前数据，以推进 PD 的自体 CRT。我们从另外三名 PD 患者 （PD02、PD03 和 PD04） 的活检成纤维细胞中生成了多个 hiPSC 系，并在良好实验室规范 （GLP） 和非 GLP 条件下使用来自临床级 hiPSC 系的 mDAC 进行了全面的安全性和有效性研究。我们还研究了扩展传代对基因组完整性和安全性的影响。根据我们的研究结果，我们提出了临床级 hiPSCs 的精细定义，并建立了 PD 中自体 CRT 的标准化释放标准。

Building on this case and FDA recommendations, we present pre-clinical data to advance autologous CRT for PD. We generated multiple hiPSC lines from biopsied fibroblasts of three additional PD patients (PD02, PD03, and PD04) and conducted comprehensive safety and efficacy studies using mDACs derived from clinical-grade hiPSC lines under both Good Laboratory Practice (GLP) and non-GLP conditions (Figure 1A). We also investigated the impact of extended passaging on genomic integrity and safety. Based on our findings, we propose a refined definition of clinical-grade hiPSCs and establish standardized release criteria for autologous CRT in PD.

来自：https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(25)00006-2