Original Article 原创文章Effective and stable gene transduction in rhesus macaque iPSCs capable of T-lineage differentiation utilizing the piggyBac system
利用 piggyBac 系统对恒河猴 iPSCs 进行有效和稳定的基因转导，可以进行 T 细胞系分化
Keywords 关键词
iPSC 恒河猴猪 BacT 细胞分化
Abbreviations 缩写
bFGF 基础成纤维细胞生长因子 CAR 嵌合抗原受体 CRISPR 聚集规律间隔短回文重复序列 EmGFP 翡翠绿荧光蛋白 HIV 人类免疫缺陷病毒 HLA 人类白细胞抗原 HSPC 造血干细胞和祖细胞 iPSC 诱导多能干细胞 ITR 倒置重复序列 MEF 小鼠胚胎成纤维细胞 NHP 非人灵长类动物 qPCR 定量聚合酶链反应 Rh-iPSC 恒河猴 iPSCRT-PCR 逆转录 PCR 艾滋病病毒 TCR T 细胞受体 tEGFR 截短的人表皮生长因子受体 VEGF 血管内皮生长因子
1. Introduction 1. 引言
Chimeric antigen receptor (CAR)-transduced T cells are effective against cancer [1]. Expanding T-cell therapy requires the development of “off-the-shelf” T-cell sources. Our previous study showed that CAR-transduced induced pluripotent stem cells (iPSCs) are expandable and can differentiate into functional T cells to suppress tumor cells in vitro and in vivo [[2], [3], [4], [5], [6]]. Furthermore, studies have demonstrated that the use of human leukocyte antigen (HLA)-edited iPSCs can effectively reduce both cost and time required for vein-to-vein manufacturing. These modified cells are capable of evading allogeneic immune rejection, and the inclusion of immune tolerance-inducing proteins such as CD47 and HLA-E can assist in developing hypoimmunogenic universal iPSCs [7,8].
嵌合抗原受体（CAR）转导的 T 细胞对癌症有效[1]。扩展 T 细胞治疗需要开发“即插即用”的 T 细胞来源。我们先前的研究表明，CAR 转导的诱导多能干细胞（iPSCs）可扩展并可分化为功能性 T 细胞，以抑制体外和体内的肿瘤细胞[[2]，[3]，[4]，[5]，[6]]。此外，研究表明使用经人类白细胞抗原（HLA）编辑的 iPSCs 可以有效降低从静脉到静脉制造所需的成本和时间。这些修改的细胞能逃避异基因免疫排斥，并且包括免疫耐受诱导蛋白如 CD47 和 HLA-E 可以协助开发低免疫原性的通用 iPSCs[7,8]。
To evaluate the safety and function of genetically modified iPSC-derived T cells, preclinical studies have used immunodeficient mouse models [6,9]. However, owing to significant differences in their characteristics compared to humans, accurate predictions cannot be achieved [10].
为评估基因改造 iPSC 衍生 T 细胞的安全性和功能，临床前研究使用了免疫缺陷小鼠模型[6,9]。然而，由于其特性与人类存在显著差异，无法实现准确的预测[10]。
Non-human primate (NHP) models such as rhesus macaques are highly similar to humans in size, lifespan, and immune system, rendering them superior models for preclinical studies [11]. In the gene editing of rhesus macaque iPSCs, knockout of specific genes using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has been reported [12,13]. However, studies of efficient gene transfer methods are limited. Human immunodeficiency virus (HIV)-based lentiviral vectors are often used for gene transfer into human iPSCs [[4], [5], [6],14]. Although they can penetrate rhesus macaque cells, gene transduction cannot be established due to the presence of HIV resistance factors such as TRIM5a [15,16]. Moreover, there is a concern regarding the potential for unforeseen carcinogenesis in transgenic cells due to the ease with which genomic integrated viral vectors can be incorporated into proto-oncogenes or their promoter regions [17]. Also, retroviral transduction into primary rhesus macaque cells has been documented in various cell types such as T cells and hematopoietic stem and progenitor cells (HSPCs) [18,19]. This technique has been particularly employed in generating iPSCs from rhesus macaque fibroblasts [20]. However, it has been noted that in pluripotent stem cells, the silencing of retrovirus DNAs occurs rapidly [21,22].
非人灵长类动物 (NHP) 模型，如恒河猴，在体型、寿命和免疫系统方面与人类非常相似，因此成为优越的临床前研究模型。在恒河猴 iPSC 的基因编辑中，利用 CRISPR/Cas9 系统敲除特定基因已被报道。在高效基因转移方法的研究中存在一定的限制。人类免疫缺陷病毒 (HIV) 基础的慢病毒载体通常被用于向人类 iPSC 进行基因转移。尽管它们可以渗透恒河猴细胞，但由于存在 HIV 抗性因子如 TRIM5a，基因导入无法建立。此外，由于基因组整合的病毒载体易于并入原癌基因或其启动子区域，因此转基因细胞可能会出现无法预测的癌变潜力。此外，已记录将逆转录病毒转导到多种恒河猴细胞类型，如 T 细胞和造血干细胞 (HSPCs)。该技术尤其被用于从恒河猴成纤维细胞产生 iPSC。然而，也有注意到在多能干细胞中，逆转录病毒 DNA 的沉默速度很快。
The piggyBac transposon vector is a virus-independent gene delivery system that uses electroporation with a transposase vector. The advantages of the piggyBac transposon vector are that it can introduce large genes and because this system is virus-free, it is safe and easy for the operator to perform experiments [23]. Cell lines, including human iPS, have the ability to efficiently edit genes using the piggyBac transposon vector [24]. Additionally, the piggyBac transposon vector has been used to generate iPSCs from rhesus macaque fibroblasts [25].
piggyBac 跳跃子载体是一种独立于病毒的基因传递系统，使用转座酶载体进行电穿孔。 piggyBac 跳跃子载体的优点是它可以引入大基因，因为该系统无病毒，对操作者来说是安全且易于执行实验。细胞系，包括人类 iPS，能够使用 piggyBac 跳跃子载体高效地编辑基因。此外，piggyBac 跳跃子载体已用于从恒河猴成纤维细胞中产生 iPSCs。
We believe that the piggyBac transposon vector gene editing system could be a new gene transduction tool for rhesus macaque iPSCs (Rh-iPSCs).
我们相信 piggyBac 跳跃子载体基因编辑系统可能成为恒河猴 iPSCs（Rh-iPSCs）的新基因转导工具。
In this study, we transduced marker genes into Rh-iPSCs using the piggyBac transposon vector/transposase vector system and generated Rh-iPSCs stably expressing these markers.
在这项研究中，我们使用 piggyBac 跳跃子载体/转座酶载体系统将标记基因转导到 Rh-iPSCs，并生成稳定表达这些标记的 Rh-iPSCs。
We selected two reporter proteins: emerald green fluorescent protein (EmGFP), which is frequently used for in vitro imaging [26], and truncated human epidermal growth factor receptor (tEGFR), which has low immunogenicity and is used as a transplanted cell marker [27].
我们选择了两种报告蛋白：常用于体外成像的祖母绿荧光蛋白（EmGFP），以及具有低免疫原性且用作移植细胞标记的截短的人表皮生长因子受体（tEGFR）。
2. Materials and methods 2. 材料和方法
2.1. Cell culture 2.1. 细胞培养
Rh-iPSC lines R1863 and R1887 were generated from T cells using a Sendai virus vector harboring KLF2, OCT3/4, SOX2, and c-MYC, as previously described [12]. Rh-iPSCs were maintained in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (Sigma-Aldrich, MO, USA) with 20% KnockOut serum replacement (Thermo Fisher Scientific, MA,USA), 1% l-Glutamine–penicillin–strepto-mycin solution (PSG, Sigma-Aldrich), MEM non-essential amino acids solution ( × 1) (Wako, Osaka, Japan), 5 ng/mL basic fibroblast growth factor (bFGF) (Wako), 10 mM 2-mercaptoethanol (Thermo Fisher Scientific), 3 μM CHIR99021 (TOCRIS bioscience, Bristol, UK), and 2 μM PD0325901 (Wako) with mouse embryonic fibroblasts (MEFs) as feeder cells.
Rh-iPSC 系列 R1863 和 R1887 是使用携带 KLF2、OCT3/4、SOX2 和 c-MYC 的仙台病毒载体从 T 细胞中产生的，如先前描述的那样。Rh-iPSCs 在 Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham（Sigma-Aldrich, MO, USA）中维持，含有 20%的 KnockOut 血清替代物（Thermo Fisher Scientific，MA, USA），1%的 L-谷氨酰胺/青霉素/链霉素溶液（PSG，Sigma-Aldrich），MEM 非必需氨基酸溶液（× 1）（和光，日本大阪），5 ng/mL 碱性成纤维母细胞生长因子（bFGF）（和光），10 mM 2-巯基乙醇（Thermo Fisher Scientific），3 μM CHIR99021（TOCRIS 生物科学，英国布里斯托），和 2 μM PD0325901（和光），以及以小鼠胚胎成纤维细胞（MEFs）作为饲养层。
2.2. PiggyBac transposon vector electroporation
2.2. PiggyBac 转座子载体电穿孔
The piggyBac transposon vector was purchased from VectorBuilder. pHL-EF1a-hcPBase-A was kindly provided by Dr. Hotta (CiRA, Kyoto, Japan). Rh-iPSCs were previously treated with 10 μM Y-27632 (Wako) for 1 h and then Rh-iPSCs were dissociated with Trypsin-EDTA solution (Sigma-Aldrich) into single cells. Single-cell suspension with 5 μg piggyBac transposon vector and 2 μg pHL-EF1a-hcPBase-A were electroporated by Maxcyte ExPERT ATx.
piggyBac 转座子载体从 VectorBuilder 购买。pHL-EF1a-hcPBase-A 由酒井贤实博士（CiRA，日本京都）提供。Rh-iPSCs 先前用 10 μM Y-27632（和光）处理 1 h，然后 Rh-iPSCs 用 Trypsin-EDTA 溶液（Sigma-Aldrich）分离成单个细胞。单细胞悬液与 5 μg piggyBac 转座子载体和 2 μg pHL-EF1a-hcPBase-A 由 Maxcyte ExPERT ATx 电穿孔。
2.3. Flow cytometry and antibodies
流式细胞术和抗体
The following antibodies were used in this study.
本研究中使用以下抗体。
The anti CD3 (SP34-2), CD34 (563), NHP CD45 (D058-1283) antibodies were purchased from BD Biosciences (New Jersey, USA). The CD4 (OKT4), and EGER (AY13) antibodies were purchased from BioLegend (San Diego, CA). CD8β (SIDI8BEE) antibody was purchased from Invitrogen (Carlsbad, CA).
抗 CD3（SP34-2）、CD34（563）、NHP CD45（D058-1283）抗体由 BD Biosciences（美国新泽西州）购买。CD4（OKT4）和 EGER（AY13）抗体由 BioLegend（美国圣地亚哥）购买。CD8β（SIDI8BEE）抗体由 Invitrogen（美国加利福尼亚州卡尔斯巴德）购买。
For cell staining, we used a BD FACSAria II or BD LSRFortessa (BD Biosciences). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Single cells were analyzed by doublet discrimination, and dead cells were depleted by propidium iodide (Sigma-Aldrich) staining.
用于细胞染色，我们使用了 BD FACSAria II 或 BD LSRFortessa（BD Biosciences）。数据使用 FlowJo 软件（Tree Star，Ashland，OR，USA）进行了分析。单个细胞通过双体鉴别进行分析，并通过使用丙啶碘化物（Sigma-Aldrich）染色来去除死细胞。
2.4. Immunofluorescence staining
2.4. 免疫荧光染色
Rh-iPSCs were fixed with 4% paraformaldehyde phosphate buffer solution (Wako). The cells were then stained with an anti-TRA-1-60 antibody (clone TRA-1-60, Millipore, MA, USA) or anti-SSEA-4 antibody (clone 813-70, Santa Cruz Biotechnology, TX, USA). The secondary antibody used was goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor™ 488 (Invitrogen). For nuclear staining, we used VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, CA, USA). Images were captured with fluorescence microscopy BZ-X810 (KEYENCE, Osaka, Japan).
Rh-iPSCs 用 4％对映甲醛磷酸盐缓冲溶液（Wako）固定。细胞然后使用抗-TRA-1-60 抗体（克隆 TRA-1-60，密理波尔，MA，USA）或抗-SSEA-4 抗体（克隆 813-70，Santa Cruz Biotechnology，TX，USA）进行染色。使用的二抗是山羊抗小鼠 IgG（H + L）高度交叉吸附的二抗，Alexa Fluor™ 488（Invitrogen）。用于核染色，我们使用了含 DAPI 的 VECTASHIELD 抗褪色封装介质（Vector Laboratories，CA，USA）。图像使用荧光显微镜 BZ-X810（基恩士，大阪，日本）捕获。
2.5. Reverse transcription PCR (RT-PCR)
2.5. 逆转录 PCR（RT-PCR）
We generated complementary DNA from Rh-iPSCs using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). TaKaRa Ex Taq (TaKaRa Bio, Shiga, Japan) was used for RT-PCR. The bands were detected using a WSE-5400-CyP Printgraph Classic (ATTO, Tokyo, Japan).
我们使用 High-Capacity cDNA Reverse Transcription Kit（Thermo Fisher Scientific）从 Rh-iPSCs 生成互补 DNA。使用 TaKaRa Ex Taq（TaKaRa Bio，Shiga，日本）进行 RT-PCR。使用 WSE-5400-CyP Printgraph Classic（ATTO，东京，日本）检测带。
2.6. Quantitative real-time PCR (qPCR)
2.6. 定量实时 PCR（qPCR）
Genomic DNA was extracted from Rh-iPSCs using NucleoSpin Tissue (TaKaRa Bio) according to the manufacturer's protocol. For the qPCR assay, we referred to previous report [28]. qPCR was performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as a reference gene and quantified using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, Osaka, Japan). We used these primers: HPRT1 Forward Primer: 5′-TTATGGACAGGACTGAACGTCTTG-3′ and HPRT1 Reverse Primer: 5′-GCACACAGAGGGCTACAATGTG-3′.
从 Rh-iPSCs 中提取基因组 DNA，使用 NucleoSpin Tissue (TaKaRa Bio) 并按照制造商的方案操作。将参考以前的报道[28]进行 qPCR 分析。使用 StepOnePlus 实时 PCR 系统 (Thermo Fisher Scientific) 进行 qPCR。以次黄嘌呤核糖转移酶 1 (HPRT1) 作为参考基因，并用 THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, 日本) 进行定量。我们使用以下引物：HPRT1 正向引物: 5′-TTATGGACAGGACTGAACGTCTTG-3′ 和 HPRT1 反向引物: 5′-GCACACAGAGGGCTACAATGTG-3′。
To measure the 5’ inverted terminal repeat (ITR) region, we used THUNDERBIRD Probe qPCR Mix (TOYOBO). To make a standard curve, we used a serial dilution of plasmid (108, 107, 106, 105, 104, 103, 102 and 101 copies/μL).
为了测量 5’倒置重复序列 (ITR) 区域，我们使用了 THUNDERBIRD 探针 qPCR Mix (TOYOBO)。为了制作标准曲线，我们使用了质粒的序列稀释 (10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 和 10 1 copies/μL)。
Vector copy number (VCN) was calculated by following formula: VCN per cell = qPCR copy number/μL of vector target/qPCR copy number/μL of reference target × 2.
向量拷贝数 (VCN) 可以通过以下公式计算：每个细胞的 VCN = 向量目标 qPCR 拷贝数/μL/参考目标 qPCR 拷贝数/μL × 2。
2.7. Teratoma formation 2.7. 胚瘤形成
2 × 106 Rh-iPSCs were suspended with 100 μL Corning Matrigel Basement Membrane Matrix (Corning, NY, USA) and 100 μL of cold D-PBS(−) (Nacalai Tesque, Kyoto, Japan). Rh-iPSCs were injected subcutaneously into the back just above the hind limb of 6-week-old female NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. The teratomas were dissected 8–10 weeks later, fixed with 10% formalin (Wako), and processed for hematoxylin and eosin staining. Images were captured with fluorescence microscopy BZ-X810 (KEYENCE).
取 2 × 10 6 Rh-iPSCs，与 100 μL Corning Matrigel Basement Membrane Matrix (Corning, 美国纽约) 和 100 μL 冷的 D-PBS(−) (Nacalai Tesque, 日本京都) 悬浮。Rh-iPSCs 被注射到 6 周大的女性 NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG)小鼠的后腿上方皮下。8-10 周后解剖胚瘤，用 10%甲醛 (Wako) 固定，进行苏木精和伊红染色。图像由荧光显微镜 BZ-X810 (KEYENCE) 捕获。
2.8. In vitro T-Cell lineage differentiation from Rh-iPSCs via HSPCs
2.8. 通过 HSPCs 从 Rh-iPSCs 体外 T 细胞谱系分化
To obtain HSPCs, Rh-iPSCs were differentiated as previously reported [12]. In brief, small clumps of Rh-iPSCs were co-cultured on C3H10T1/2 feeder cells with Sac medium (Iscove's Modified Dulbecco's Medium (Sigma-Aldrich) consisting 15% fetal bovine serum (FBS), 1% PSG, insulin–transferrin–selenium (ITS-G) (1X) (Gibco), 450 mM monothioglycerol (Nacalai Tesque), 50 μg/mL ascorbic acid (Nacalai Tesque), in addition to 20 ng/mL vascular endothelial growth factor (VEGF) (R&D Systems, MN, USA) for 7 days. After 7 days, the cells were cultured in Sac medium supplemented with 30 ng/mL stem cell factor (SCF) (R&D Systems) and 10 ng/mL recombinant human Flt3-ligand (rhFlt3-L PeproTech, NJ, USA). After 14 days of culture, we obtained HSPCs from Sac-like structures derived from iPSCs.
获取 HSPCs，Rh-iPSCs 按照先前的报告进行分化[12]。简而言之，Rh-iPSCs 小团块在 C3H10T1/2 饲养细胞上与 Sac 培养基（含 15%胎牛血清（FBS）、1%PSG、胰岛素-转铁蛋白-硒（ITS-G）（1X）450 mM 巯基甘油（Nacalai Tesque）、50μg/mL 抗坏血酸（Nacalai Tesque），以及 20 ng/mL 血管内皮生长因子（VEGF）（R&D Systems，MN，USA））共培养 7 天。7 天后，细胞在 Sac 培养基中培养，补充 30 ng/mL 干细胞因子（SCF）（R&D Systems）和 10 ng/mL 重组人源 Flt3 配体（rhFlt3-L PeproTech，NJ，USA）。培养 14 天后，从 iPSCs 衍生的类似 Sac 结构中获得了 HSPCs。
Next, we transferred HSPCs onto OP9-DL1 cells and co-cultured in OP9 medium (aMEM consisting 15% FBS, 1% PSG, ITS-G (1X), 50 μg/mL ascorbic acid (Nacalai Tesque)) supplemented with 1 ng/mL recombinant human IL-7 (PeproTech) and 10 ng/mL rhFlt3-L. After 21 days culturing, we obtained CD8β, CD4 double positive T cells (DP cells).
然后，我们将 HSPCs 转移到 OP9-DL1 细胞上，在 OP9 培养基（aMEM 包含 15% FBS、1%PSG、ITS-G（1X）、50μg/mL 抗坏血酸（Nacalai Tesque））中添加 1 ng/mL 重组人源 IL-7（PeproTech）和 10 ng/mL rhFlt3-L 共培养。培养 21 天后，我们获得了 CD8β、CD4 双阳性 T 细胞（DP 细胞）。
2.9. Statistics 2.9. 统计学
All statistical analyses were performed using the Prism software (GraphPad Software, CA, USA). In Fig. 5c and e, a two-sided Student's t-test was used to compare the two groups in parametric data. P > 0.05 was considered not significant.
所有统计分析均使用 Prism 软件（GraphPad Software，CA，USA）进行。在图 5c 和 e 中，使用双侧 Student's t 检验比较参数数据中的两组。P > 0.05 被认为不显著。
3. Results 结果
3.1. Gene transduction utilizing piggyBac transposon vector enables long-term gene expression in Rh-iPSCs while maintaining undifferentiated markers
3.1. 利用 piggyBac 转座子载体进行基因转导，实现 Rh-iPSCs 中长期基因表达并保持未分化标记物
First, the piggyBac transposon vector carrying EmGFP as a fluorescent marker and the piggyBac transposase-expressing vector pHL-EF1a-hcPBase-A were transduced into Rh-iPSCs (Fig. 1a). Three days after gene transduction, the expression of EmGFP was analyzed by fluorescence microscopy and flow cytometry, and approximately 40% of the iPSCs expressed GFP (Fig. 1b, c, d).
首先，在 Rh-iPSCs 中转导携带 EmGFP 作为荧光标记物的 piggyBac 转座子载体和表达 piggyBac 转座酶的载体 pHL-EF1a-hcPBase-A（图 1a）。基因转导后 3 天，通过荧光显微镜和流式细胞术分析 EmGFP 的表达，约 40%的 iPSCs 表达 GFP（图 1b、c、d）。
Fig. 1. Flow chart for gene transduction into Rh-iPSCs and expression of EmGFP. a Schematic constructs of the piggyBac transposon vector and transposase-expressing vector. ITR: inverted terminal repeat, CAG: promoter. b Scheme of electroporation using the piggyBac system. Rh-iPSCs were dissociated into single cells and transduced with the piggyBac transposon and transposase-expressing vectors using a MaxCyte ATx electroporation device. Three days after electroporation, the cells were observed by fluorescence, and 7 days after electroporation, they were analyzed by flow cytometry. c Rhesus macaque iPSC line R1863 was transduced with a piggyBac transposon vector expressing EmGFP and a transposase-expressing vector by electroporation. After 3 days, EmGFP expression was observed using fluorescence microscopy. The scale bar indicates 200 μm. d Flow cytometry analysis of EmGFP expression 7 days after electroporation. The EmGFP-positive cells were sorted and cultured for several weeks.
图 1. Rh-iPSCs 基因载入和 EmGFP 表达的流程图。a 跳跃子体素载体和表达跳跃酶的载体的示意结构。ITR：反向末端重复序列，CAG：启动子。b 使用跳跃子体系统的电穿孔示意图。Rh-iPSCs 被分散为单个细胞，并使用 MaxCyte ATx 电穿孔装置转导了跳跃子体素和表达跳跃酶的载体。电穿孔后三天，细胞通过荧光观察，电穿孔后 7 天，通过流式细胞术分析。c 长尾猴 iPSC 系 R1863 通过电穿孔转导了表达 EmGFP 的跳跃子体载体和表达跳跃酶的载体。3 天后，用荧光显微镜观察 EmGFP 表达。比例尺为 200μm。d 电穿孔后 7 天 EmGFP 表达的流式细胞术分析。EmGFP 阳性细胞被分选并培养数周。
To purify transduced Rh-iPSCs, GFP-positive cells were sorted and cultured. Compared to the original iPSCs, almost all transgenic iPSCs were GFP-positive and they stably expressed GFP, even after 4 weeks (Fig. 2a). Gene transfer with the piggyBac transposon vector had no noticeable effect on the morphology of the transduced iPSCs or the undifferentiated cell surface markers SSEA4 and TRA-1-60 (Fig. 2b). Transcription factors Nanog, KLF4, POU5F1, SOX2, and c-Myc are mainly expressed in undifferentiated iPSCs. We examined the expression of these genes in transgenic and parental iPS using reverse transcription PCR (RT-PCR). Gene-transduced Rh-iPSCs and parental Rh-iPSCs maintained transcriptional expression.
为了纯化转导的 Rh-iPSCs，筛选和培养了 GFP 阳性细胞。与原始 iPSCs 相比，几乎所有转基因 iPSCs 均为 GFP 阳性，它们在 4 周后仍然稳定表达 GFP（图 2a）。使用跳跃子体载体进行基因转移对转导 iPSCs 的形态或未分化细胞表面标记 SSEA4 和 TRA-1-60 没有明显影响（图 2b）。转录因子 Nanog、KLF4、POU5F1、SOX2 和 c-Myc 主要在未分化 iPSCs 中表达。我们通过逆转录 PCR（RT-PCR）检查转基因和父级 iPS 中这些基因的表达。基因转导的 Rh-iPSCs 和父级 Rh-iPSCs 都保持转录表达。
Fig. 2. Character of EmGFP-transduced Rh-iPSCs by the piggyBac system. a EmGFP expression in sorted Rh-iPSCs The sorted Rh-iPSCs maintained EmGFP expression for 4 months at least. b Expression of undifferentiated markers SSEA4 and TRA-1-60 by immunofluorescence staining. The scale bar indicates 200 μm. c Comparison of pluripotency gene expression by RT-PCR between parental Rh-iPSCs and sorted EmGFP-transduced Rh-iPSCs
图 2. 通过 piggyBac 系统转导的 EmGFP 修饰的 Rh-iPSCs 的特性。 a 胚胎干细胞 Rh-iPSCs 的 EmGFP 表达 排序后的 Rh-iPSCs 至少保持 4 个月的 EmGFP 表达。 b 通过免疫荧光染色观察到未分化标记物 SSEA4 和 TRA-1-60 的表达。比例尺表示 200 微米。 c 通过 RT-PCR 比较母源 Rh-iPSCs 和排序后的 EmGFP 修饰的 Rh-iPSCs 之间的多能基因表达
Next, we generated a piggyBac transposon vector expressing tEGFR and attempted to transduce it into iPSCs (Fig. 3a). tEGFR was efficiently transfected into two Rh-iPSC clones (Fig. 3b). Sorting of cells with high tEGFR expression in each clone showed that tEGFR was still stably expressed after 14 weeks (Fig. 3c). The expression of Nanog, KLF4, POU5F1, SOX2, and c-Myc was confirmed by RT-PCR and was maintained after gene transfer (Fig. 3d). We named these Rh-iPSCs as tEGFR-Rh-iPS cells.
接下来，我们构建了一个表达 tEGFR 的 piggyBac 转座子载体，并尝试将其转导到 iPSC 中（图 3a）。 tEGFR 被高效转染到了两个 Rh-iPSC 克隆中（图 3b）。在每个克隆中筛选出 tEGFR 表达高的细胞后，结果显示 tEGFR 在 14 周后仍然稳定表达（图 3c）。通过 RT-PCR 确认 Nanog、KLF4、POU5F1、SOX2 和 c-Myc 的表达，并在基因转移后得以保持（图 3d）。我们将这些 Rh-iPSC 命名为 tEGFR-Rh-iPS 细胞。
Fig. 3. tEGFR gene transduction into Rh-iPSCs by the piggyBac system. a Schematic constructs of the piggyBac transposon vector expressing tEGFR. b Successful gene transduction in Rh-iPSC lines. tEGFR expression was analyzed using flow cytometry. tEGFR high expressing populations were sorted and cultured. c tEGFR expression in tEGFR-Rh-iPSCs The tEGFR-Rh-iPSC lines, R1863-tEGFR and R1887-tEGFR, maintained exogenous gene expression for 14 weeks at least. d Analysis of pluripotency genes by RT-PCR. e Transgene copy number of integration by the piggyBac system. f Teratoma formation assay. tEGFR-Rh-iPSCs differentiated into all three germ layers. GE, gut-like epithelium; C, cartilage; NC, neural crest. Scale bars indicate 100 μm.
将 tEGFR 基因通过 piggyBac 系统转导到 Rh-iPSCs 中。a piggyBac 转座子表达 tEGFR 的示意图。b Rh-iPSC 系列中成功的基因转导。使用流式细胞术分析 tEGFR 表达。tEGFR 高表达群体被分选并培养。c tEGFR 在 tEGFR-Rh-iPSCs 中的表达。tEGFR-Rh-iPSC 系列 R1863-tEGFR 和 R1887-tEGFR，至少维持 14 周的外源基因表达。d 通过 RT-PCR 分析多能基因。e piggyBac 系统整合的转基因拷贝数。f Teratoma 形成试验。tEGFR-Rh-iPSC 分化为所有三个胚层。GE，类肠状上皮；C，软骨；NC，神经嵴。标尺表示 100 微米。
Fig. 4. The differentiation into T-cell lineage cells from tEGFR-Rh-iPSCs. a Schematic illustration of differentiation from Rh-iPSCs to HSPCs. b Flow cytometric analysis of differentiated HSPCs based on CD34 and CD45 expression. HSPCs from tEGFR-Rh-iPSCs maintained tEGFR expression. c Comparison of the surface antigen profiles of HPCs. Each dot represents one biological replicate (n = 6). Non-paired Student's t-test. ns: not significant. d Schematic illustration of differentiation from HSPCs to T-cell lineage cells. e Flow cytometric analysis of differentiated T-cell lineage cells. f Comparison of the surface antigen profiles of DP cells. Each dot represents one biological replicate (n = 6). Non-paired Student's t-test. ns: not significant.
图。4. T 细胞系细胞从 tEGFR-Rh-iPSC 分化出来。a 从 Rh-iPSC 分化为 HSPCs 的示意图。b 根据 CD34 和 CD45 表达的不同，对分化 HSPCs 进行流式细胞术分析。来自 tEGFR-Rh-iPSC 的 HSPCs 保持 tEGFR 表达。c HPCs 表面抗原分布的比较。每个点代表一个生物重复（n = 6）。未配对的学生 t 检验。ns：不显著。d 从 HSPCs 分化为 T 细胞系细胞的示意图。e 分化的 T 细胞系细胞的流式细胞术分析。f DP 细胞的表面抗原分布的比较。每个点代表一个生物重复（n = 6）。未配对的学生 t 检验。ns：不显著。
The copy number of tEGFR was measured by qPCR for each clone, and approximately one copy insertion was confirmed (Fig. 3e).
每个克隆体的 tEGFR 拷贝数通过 qPCR 测定，确认了大约有一个拷贝插入（图 3e）。
Taken together, Rh-iPSCs transduced with the piggyBac transposon vector maintained long-term and stable expression of transgenes while maintaining the expression of cell surface undifferentiated markers and transcription factors.
总的来说，用 piggyBac 跳跃子转座子载体转导的 Rh-iPSCs 在保持细胞表面未分化标记物和转录因子表达的同时，保持了长期稳定的基因表达。
3.2. Gene-transduced Rh-iPSCs form teratomas
3.2. 转基因 Rh-iPSCs 形成畸胎瘤。
Untransfected Rh-iPSCs have been shown to form teratomas in vivo and to have pluripotency [12]. To test the pluripotency of tEGFR-Rh-iPSCs in vivo, we subcutaneously injected them into immunodeficient mice and observed their ability to form teratomas. The injected cells formed teratomas after 8 weeks. Histological examination revealed endodermal, mesodermal, and ectodermal tissues (Fig. 3f).
未转染的 Rh-iPSCs 已被证实在体内形成畸胎瘤并具有多能性[12]。为了测试 tEGFR-Rh-iPSCs 在体内的多能性，我们将它们皮下注射到免疫缺陷小鼠中，并观察其形成畸胎瘤的能力。注射的细胞在 8 周后形成了畸胎瘤。组织学检查显示形成了内胚层、中胚层和外胚层组织（图 3f）。
3.3. tEGFR-Rh-iPSCs maintain their expression after HSPCs differentiation
3.3. tEGFR-Rh-iPSCs 在 HSPCs 分化后保持其表达
During iPSC differentiation, exogenous genes may be silenced through epigenetic changes. To test whether tEGFR-Rh-iPSCs could maintain tEGFR expression after differentiation into hematopoietic cells, we differentiated them into HSPCs with the support of 10T1/2 feeder cells (Fig. 4a). tEGFR-Rh-iPSCs differentiated into CD34-positive cells and parental cells, and maintained tEGFR expression (Fig. 4b, c).
在 iPSC 分化过程中，外源基因可能通过表观遗传学变化被沉默。为了测试 tEGFR-Rh-iPSCs 在分化为造血细胞后能否保持 tEGFR 的表达，我们将它们与 10T1/2 供体细胞一起分化为 HSPCs（图 4a）。tEGFR-Rh-iPSCs 分化为 CD34 阳性细胞和母细胞，并保持了 tEGFR 的表达（图 4b，c）。
3.4. tEGFR-Rh-iPSCs maintained tEGFR expression after differentiation into T cells
3.4. tEGFR-Rh-iPSCs 在分化为 T 细胞后保持了 tEGFR 的表达
HSPCs derived from tEGFR-Rh-iPSCs were differentiated into CD8/CD4 double positive thymocytes (DP cells) (Fig. 4d). Both tEGFR-Rh-iPSCs and parental Rh-iPSCs differentiated into CD8b+/CD4+/CD3+/CD45+ cells and DP cells from tEGFR-Rh-iPSCs maintained tEGFR expression (Fig. 4e, f).
从 tEGFR-Rh-iPSCs 分化出的 HSPCs 分化为 CD8/CD4 双阳性胸腺细胞（DP 细胞）（图 4d）。tEGFR-Rh-iPSCs 和母 Rh-iPSCs 都分化为 CD8b+/CD4+/CD3+/CD45+细胞和 tEGFR-Rh-iPSCs 的 DP 细胞保持了 tEGFR 的表达（图 4e，f）。
These results suggest that tEGFR gene transfer with the piggyBac system does not affect the T-lineage differentiation of iPSCs.
这些结果表明，利用 piggyBac 系统对 tEGFR 基因进行转移不会影响 iPSCs 的 T 细胞系分化。
4. Discussion 讨论
In this study, we established a gene transduction method using a piggyBac system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes.
在这项研究中，我们建立了使用 piggyBac 系统的基因转导方法，证明在 Rh-iPSCs 中具有高效率和稳定的转基因表达。这些 iPSC 能够分化为表达转基因的造血干细胞和 T 细胞。
The piggyBac system is easy to manufacture as it is virus-free and requires less time than viral transduction. There is a concern regarding the potential for unforeseen carcinogenesis in transgenic cells due to the ease with which viral vectors are incorporated into proto-oncogenes or their promoter regions [29]. Analysis of gene insertion sites using the piggyBac system showed that the frequency of insertion around transcription start sites and CpG islands was significantly lower than that of retroviral vectors [29]. Additionally, the frequency of insertion into the genomic safe harbor is considerably higher than that of lentiviruses [29].
piggyBac 系统易于制造，因为它无病毒且比病毒转导需要更少的时间。由于病毒载体很容易被整合到原癌基因或其启动子区域中，对转基因细胞中未预期的癌变的潜在风险存在一定担忧。使用 piggyBac 系统进行基因插入位点分析显示，插入位点周围转录起始位点和 CpG 岛的频率明显低于逆转录病毒载体的频率。此外，插入到基因组安全定位的频率比 HIV 载体显著更高。
In the clinical application of iPSC-based medicines, it is necessary to predict their safety because unexpected side effects can occur and they are sometimes lethal to patients. This outcome is attributed to the preclinical study model conducted on mice considering the substantial differences in immune system and body size between humans and mice [30].
在 iPSC 药物的临床应用中，需要预测它们的安全性，因为可能会出现意想不到的副作用，有时对患者致命。这种结果归因于在小鼠上进行的临床前研究模型，考虑到人类和小鼠之间的免疫系统和体型巨大差异。
Rhesus macaques serve as valuable primate model animals in preclinical studies in humans owing to their anatomical and physiological similarities to humans [10,11,31]. Rhesus macaque iPSCs have been successfully generated from fibroblasts [20] and T cells [12] and differentiated into immune cells [12,32].
恒河猴作为重要的灵长类模式动物在人类临床前研究中发挥着重要作用，因为其解剖和生理与人类相似。恒河猴 iPSCs 成功从成纤维细胞和 T 细胞中培育出来，并分化为免疫细胞。
For clinical application of human iPSC-derived cells, it is necessary to produce homogeneously differentiated cells from genetically engineered iPSCs. Genome editing using the CRISPR-Cas9 system and gene transfer using lentiviral vectors are being practiced [5,14]. In previous studies on Rh-iPSCs, genome editing was performed using the CRISPR-Cas9 system [12,13]; however, gene transduction by lentiviral vectors has not been reported. We attempted gene transduction into Rh-iPSCs with a HIV-based lentiviral vector but were unable to confirm gene transfer. This could be attributed to the resistance of rhesus macaque cells toward HIV infection [15,16]. There are some improved HIV-based lentiviral vectors and Simian immunodeficiency virus (SIV)-based lentiviral vectors, but they are not easy to access [16,33]. Moreover, lentiviral vector transduction of HSPCs in rhesus macaques has been suggested to induce aberrant clonal hematopoiesis [34].
为了临床应用人类 iPSC 衍生细胞，需要从基因工程的 iPSC 中产生同质分化的细胞。使用 CRISPR-Cas9 系统进行基因组编辑和使用慢病毒载体进行基因转移已在实践中应用[5,14]。在之前的 Rh-iPSC 研究中，使用 CRISPR-Cas9 系统进行了基因组编辑[12,13]; 然而，利用慢病毒载体进行基因转导尚未报道。我们尝试使用基于 HIV 的慢病毒载体将基因转导到 Rh-iPSC 中，但未能确认基因转移。这可能是由于恒河猴细胞对 HIV 感染的抵抗力[15,16]。已经有一些改进的基于 HIV 的慢病毒载体和猴类免疫缺陷病毒（SIV）基础的慢病毒载体，但不易获得[16,33]。此外，已经有建议表明，在恒河猴的 HSPCs 中进行慢病毒载体转导可能会诱导异常的克隆性造血[34]。
Therefore, we used the piggyBac transposon vector as a simple method for gene transfection into Rh-iPSCs. Compared to lentiviruses, the piggyBac transposon vector can introduce a large gene (>10 kbp), and because a plasmid is used, the method is simple and does not require viral containment.
因此，我们使用 piggyBac 转座子载体作为将基因转染到 Rh-iPSC 的简单方法。与慢病毒相比，piggyBac 转座子载体可以引入较大的基因(>10 kbp)，并且因使用质粒，该方法简单且无需病毒约束。
Rh-iPSCs transduced with piggyBac transposon vectors maintained high gene expression even after several months of culture and silencing did not occur (Fig. 2). Considering that piggyBac transposon vectors are used for reprogramming Rh-iPSCs, this suggests that the piggyBac transposon vector is an outstanding tool for gene introduction into rhesus macaque cells [24,25].
Rh-iPSC 经 piggyBac 转座子载体转导后即使在几个月的培养后仍保持高基因表达，且未发生沉默（图 2）。考虑到 piggyBac 转座子载体被用于重编程 Rh-iPSCs，这表明 piggyBac 转座子载体是将基因引入恒河猴细胞的出色工具[24,25]。
The teratomas assay indicated that the grafted tEGFR-Rh-iPSCs differentiated into three germ layers and showed multilineage differentiation. We did not examine tEGFR expression in any of the three germ layers. Nevertheless, validating its expression would broaden the applicability of this transduction system across various tissue types.
畸胎瘤试验表明，移植的 tEGFR-Rh-iPSCs 分化为三个胚层，并显示出多系分化。我们没有检查 tEGFR 在任何三个胚层中的表达。然而，验证其表达将扩大这种转导系统在各种组织类型中的适用性。
Gene-engineered adoptive cell therapy, typified by CAR-transduced T-cell therapy, has shown great promise as a new cancer therapy. However, there are several issues, including the difficulty in obtaining a sufficient quantity of cells for transplantation, the time-intensive manufacturing process, and the associated high costs. iPSCs have an unlimited self-renewal capacity and iPSC-derived cytotoxic T cells proliferate efficiently upon repeated stimulation. Therefore, iPSCs are expected to serve as a source of cells for T-cell therapy.
基因工程的养成细胞疗法，以 CAR 转导 T 细胞疗法为代表，作为一种新的癌症疗法展现出巨大的潜力。然而，存在一些问题，包括难以获得足够数量的用于移植的细胞、耗时的制造过程和相关的高成本。iPSCs 具有无限的自我更新能力，iPSC 衍生的细胞毒性 T 细胞在重复刺激下能够高效增殖。因此，期望 iPSCs 能够作为 T 细胞疗法的细胞来源。
We differentiated tEGFR-Rh-iPSCs into HSPCs and then into T-cell lineages. tEGFR-Rh-iPSC-derived differentiated cells maintained tEGFR expression.
我们将 tEGFR-Rh-iPSC 分化为 HSPCs，然后分化为 T 细胞系。tEGFR-Rh-iPSC 衍生的分化细胞保持了 tEGFR 的表达。
For clinical applications, iPSCs must be cultured in the absence of feeder cells and cultured and differentiated under defined component conditions. In this study, we used mouse embryonic fibroblasts (MEF) and 10T1/2 and OP9 cells originating from mouse cells. A recent report showed Rh-iPSC feeder-free generation and cultivation under chemically defined conditions [25], and our group reported that human iPSCs can differentiate into killer T cells under feeder-free and chemically defined conditions [6]. By advancing these technologies, we expect to establish an Rh-iPSC differentiation method.
对于临床应用，iPSCs 必须在无投食层细胞的条件下培养，并在定义的组分条件下培养和分化。在这项研究中，我们使用了来源于老鼠的胚胎成纤维细胞（MEF）以及 10T1/2 和 OP9 细胞。最近的报告显示 Rh-iPSC 在无投食层的情况下在化学定义条件下生成和培养，我们小组报告了在无投食层和化学定义条件下人类 iPSCs 能够分化为杀伤性 T 细胞。通过推进这些技术，我们期望建立 Rh-iPSC 分化方法。
In our future studies, we plan to generate target gene-expressing T cells derived from genetically modified Rh-iPSCs. The expected genes were CAR and endogenous T-cell receptor (TCR). Functional assays can be performed in vitro and in vivo by inducing the expression of these proteins. A previous report has shown that anti-CD20 CAR-transduced peripheral T cells cause serious adverse effects [35]. In a phase I trial of piggyBac-modified CD19 CAR-T therapy conducted in Australia piggyBac-modified CD19 CAR-T cells from transplant donors were administered to 10 patients with relapsed B-cell tumors post-hematopoietic stem cell transplantation. Although the therapy demonstrated high efficacy, with reported remission in all 10 treated patients, two patients developed CAR-T cell-derived T-cell lymphoma [36]. It was reported that the CAR-T cell-derived T-cell lymphoma cells had a relatively high CAR gene copy number (24 copies). While no specific integration into cancer gene regions was observed in the analysis of CAR gene insertion sites, integration into the BACH2 gene region, considered a tumor suppressor gene, was observed in both cases. The BACH2 gene is a transcription factor associated with cutaneous T-cell lymphoma, and its expression is suppressed in CD4-positive T cells. Since the mechanism are still unclear, it was concluded that follow-up is important [37].
我们未来的研究计划通过基因改造的 Rh-iPSCs 派生靶基因表达 T 细胞。所期望的基因是 CAR 和内源性 T 细胞受体（TCR）。功能评价可通过诱导这些蛋白质表达在体内外进行。先前的报告显示，抗 CD20 CAR 转染的外周 T 细胞引起严重不良反应。在澳大利亚进行的一个 piggyBac 改造 CD19 CAR-T 疗法的 I 期试验中，从移植供体获取的 piggyBac 改造 CD19 CAR-T 细胞被用于治疗 10 例经髓系干细胞移植后复发的 B 细胞肿瘤患者。尽管该疗法显示出高有效性，所有接受治疗的 10 名患者中都报告出现缓解，但两名患者出现了 CAR-T 细胞源性 T 细胞淋巴瘤。据报道，CAR-T 细胞源性 T 细胞淋巴瘤细胞中 CAR 基因拷贝数相对较高（24 拷贝）。尽管在 CAR 基因插入位点分析中没有观察到特异性整合到癌基因区域，但在两个案例中均观察到整合到 BACH2 基因区域，该基因被认为是肿瘤抑制基因。BACH2 基因是一个与皮肤 T 细胞淋巴瘤相关的转录因子，它的表达在 CD4 阳性 T 细胞中被抑制。由于机制尚不清楚，因此得出结论需要重点关注后续工作。
For our piggyBac-based iPSC transduction system, we are planning to conduct careful assessments, including checking the copy number and specific integration into cancer gene regions, as well as mutations in genes related to T-cell lymphoma. These analyses will help us select safe iPSC clones for further investigation. Moreover, because of the easiness of gene editing, iPS cells can carry suicide genes such as tEGFR [38]. Therefore, in the event of a serious adverse reaction, it can be expected to be eliminated immediately.
对于我们基于 piggyBac 的 iPSC 转导系统，我们计划进行仔细的评估，包括检查拷贝数和特定整合到癌基因区域，以及与 T 细胞淋巴瘤相关基因的突变。这些分析将帮助我们选择安全的 iPSC 克隆用于进一步研究。此外，由于基因编辑的便利性，iPS 细胞可以携带诸如 tEGFR [38]等自杀基因。因此，在发生严重不良反应时，可以预期立即被消除。
SIV is a model HIV that infects rhesus macaques. Research on antigen-specific TCR against SIV-expressing T cells is progressing, and effective TCRs have been cloned [39]. Therefore, we generated Rh-iPSCs that express TCR against SIV and differentiate into killer T cells. The generated T cells will be transplanted into SIV-infected rhesus macaques, and their therapeutic effect and safety will be evaluated.
酶联免疫吸附分析（enzyme-linked immunosorbent assay, ELISA）是一种常用于检测分子的方法。该方法通过对待检样品中的特定蛋白质进行定性和定量测定，来研究细胞因子水平、抗体浓度、抗原或抗体的相互作用等。
5. Conclusion 结论
The piggyBac system is a highly effective gene transfer tool for rhesus macaque iPSCs. These results are expected to substantially contribute to the advancement of preclinical studies on rhesus macaque iPSCs.
piggyBac 系统是恒河猴 iPSC 的高效基因转移工具。这些结果有望在恒河猴 iPSC 的临床前研究方面做出重大贡献。
Credit authorship contribution statement
作者贡献陈述
Masahiro Tanaka: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Roles/Writing - original draft, Writing - review & editing. Yoshihiro Iwamoto: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Visualization. Bo Wang: Investigation, Supervision, Writing - review & editing. Eri Imai: Investigation, Validation. Munehiro Yoshida: Investigation. Shoichi Iriguchi: Conceptualization, Methodology, Supervision, Writing - review & editing. Shin Kaneko: Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing.
田中正弘：概念化，数据整理，形式分析，调查，方法学，项目管理，资源，验证，可视化，角色/撰写 - 原始草稿，撰写 - 审阅和编辑。岩本良広：概念化，数据整理，形式分析，调查，方法学，项目管理，资源，可视化。王波：调查，监督，撰写 - 审阅和编辑。今井恵里：调查，验证。吉田宗弘：调查。入口祥一：概念化，方法学，监督，撰写 - 审阅和编辑。金子慎：筹资，项目管理，资源，监督，撰写 - 审阅和编辑。
Declaration of competing interest
竞争兴趣声明
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shin Kaneko is a founder, shareholder, and chief scientific officer at Thyas, Co., Ltd., and received research funding from Takeda Pharmaceutical, Co., Ltd., Kirin Holdings, Co., Ltd., Astellas, Co., Ltd, Terumo Co., Ltd., Tosoh, Co., Ltd., and Thyas, Co., Ltd.
作者声明以下可能被视为潜在竞争利益的财务利益/个人关系：金子慎是 Thyas，Co.，Ltd.的创始人，股东和首席科学官，并从武田制药有限公司，Kirin 控股有限公司，艾士特拉斯有限公司，特林公司，有限公司，东盛有限公司以及 Thyas 有限公司获得了研究资助。
Acknowledgments 致谢
We thank every laboratory staff and Ken Fukumoto from bottom of my heart.
我衷心感谢每个实验室工作人员以及福元健。
I would like to thank Drs. Hirofumi Akari (Kyoto University), Tatsuo Shioda, Emi E. Nakayama (Osaka University), Tomoyuki Miura (Kyoto University) for useful discussions.
感谢京都大学的明里宏史博士，大阪大学的塩田辰男，中山恵美，京都大学的三浦智之等对有益的讨论表示感谢。
We would like to thank Editage (www.editage.jp) for English language editing.
我们要感谢 Editage（www.editage.jp）进行英文编辑。
This study was supported by The Cooperative Research Program of the Primate Research Institute, Kyoto University, and the Cooperative Research Program (Joint Usage/Research Center Program) of the Institute for Frontier Life and Medical Sciences, Kyoto University (2020-B-69).
本研究得到了京都大学灵长类研究所合作研究计划以及京都大学前沿生命医学科学研究所的联合研究计划（2020-B-69）的支持。
References 参考文献
- [1]Chimeric antigen receptor T cells for sustained remissions in leukemia
白血病持续缓解的嵌合抗原受体 T 细胞N Engl J Med, 371 (2014), pp. 1507-1517, 10.1056/nejmoa1407222
N Engl J Med，371（2014），页码 1507-1517，doi: 10.1056/nejmoa1407222 - [2]Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation
通过重编程至多能性再分化生成年轻化的特定抗原 T 细胞Cell Stem Cell, 12 (2013), pp. 114-126, 10.1016/j.stem.2012.11.002
《细胞干细胞》，12（2013），第 114-126 页，DOI：10.1016/j.stem.2012.11.002 - [3]Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy
通过多能性重编程为养活免疫疗法生成高增殖、恢复活力的细胞毒性 T 细胞克隆Mol Ther, 29 (2021), pp. 3027-3041, 10.1016/j.ymthe.2021.05.016
Mol Ther，29（2021），pp. 3027-3041，10.1016/j.ymthe.2021.05.016 - [4]The therapeutic potential of multiclonal tumoricidal T cells derived from tumor infiltrating lymphocyte-1derived iPS cells
源自肿瘤浸润淋巴细胞-1 来源 iPS 细胞的多克隆抗肿瘤 T 细胞的治疗潜力Commun Biol, 4 (2021), pp. 1-16, 10.1038/s42003-021-02195-x
《Commun Biol》, 4 (2021), pp. 1-16, 10.1038/s42003-021-02195-x - [5]Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells
优化来源于人类诱导多能干细胞的 CAR T 细胞的增殖和持久性Nat Biomed Eng, 7 (2023), pp. 24-37, 10.1038/s41551-022-00969-0
《Nat Biomed Eng》, 7 (2023), pp. 24-37, 10.1038/s41551-022-00969-0 - [6]A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy
从 iPSCs 再生 T 细胞的一种临床适用且可扩展的方法，用于实现现货 T 细胞免疫疗法Nat Commun, 12 (2021), pp. 1-15, 10.1038/s41467-020-20658-3
Nat Commun，12（2021），第 1-15 页，10.1038/s41467-020-20658-3 - [7]Targeted disruption of HLA genes via CRISPR-cas9 generates iPSCs with enhanced immune compatibility
通过 CRISPR-cas9 靶向破坏 HLA 基因，生成具有增强免疫适应性的 iPSCs。Cell Stem Cell, 24 (2019), pp. 566-578.e7, 10.1016/j.stem.2019.02.005
Cell Stem Cell, 24 (2019), 页码 566-578.e7, 10.1016/j.stem.2019.02.005 - [8]Generation of hypoimmunogenic T cells from genetically engineered allogeneic human induced pluripotent stem cells
从基因工程的同种异体人诱导多能干细胞中生成低免疫原性 T 细胞Nat Biomed Eng, 5 (2021), pp. 429-440, 10.1038/s41551-021-00730-z - [9]Non–clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti–glypican-3 chimeric antigen receptor-expressing natural killer/innate lymphoid cells
诱导多能干细胞衍生的抗甘肽酸-3 嵌合抗原受体表达自然杀伤/先天淋巴细胞的非临床疗效、安全性和稳定的临床细胞处理Cancer Sci, 111 (2020), pp. 1478-1490, 10.1111/cas.14374
癌症科学, 111 (2020), pp. 1478-1490, 10.1111/cas.14374 - [10]Spontaneous, naturally occurring cancers in non-human primates as a translational model for cancer immunotherapy
非人灵长类动物自发性发生的癌症作为癌症免疫治疗的转化模型J Immunother Cancer, 11 (2023), 10.1136/jitc-2022-005514
免疫治疗癌症, 11 (2023), 10.1136/jitc-2022-005514 - [11]Animal models for HIV/AIDS research
用于 HIV/AIDS 研究的动物模型Nat Rev Microbiol, 10 (2012), pp. 852-867, 10.1038/nrmicro2911
微生物学评论，10（2012），第 852-867 页，10.1038/nrmicro2911 - [12]Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease
通过基因编辑的恒河猴 iPSCs 生成对病毒敏感性变化的巨噬细胞，以模拟人类疾病Mol Ther - Methods Clin Dev, 21 (2021), pp. 262-273, 10.1016/j.omtm.2021.03.008 - [13]Rhesus iPSC safe harbor gene-editing platform for stable expression of transgenes in differentiated cells of all germ layers
猕猴 iPSC 安全港基因编辑平台，用于在所有生殖层分化细胞中稳定表达转基因Mol Ther, 25 (2017), pp. 44-53, 10.1016/j.ymthe.2016.10.007 - [14]Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy
从诱导多能干细胞生成肿瘤靶向人类 T 淋巴细胞用于癌症治疗Nat Biotechnol, 31 (2013), pp. 928-933, 10.1038/nbt.2678
等位基因生态学 (Nat Biotechnol, 31 (2013), pp. 928-933, 10.1038/nbt.2678) - [15]Inhibition of HIV-1 replication by simian restriction factors, TRIM5α and APOBEC3G
通过猴限制因子 TRIM5α 和 APOBEC3G 抑制 HIV-1 复制Gene Ther, 14 (2007), pp. 185-189, 10.1038/sj.gt.3302852
《基因治疗》，14（2007），第 185-189 页，10.1038/sj.gt.3302852 - [16]Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system
使用基于猴免疫缺陷病毒的慢病毒载体系统高效地将基因转移至恒河猴再生造血干细胞Blood, 103 (2004), pp. 4062-4069, 10.1182/blood-2004-01-0045
血液，103 (2004)，第 4062-4069 页，10.1182/blood-2004-01-0045 - [17]LMO2-Associated clonal T cell proliferation in two patients after gene therapy for SCID-X1
LMO2 相关的两名患者在进行 SCID-X1 基因治疗后出现的克隆 T 细胞增殖Science, 302 (80) (2003), pp. 415-419, 10.1126/science.1088547
科学，302(80) (2003)，第 415-419 页，10.1126/science.1088547 - [18]Adoptive transfer of engineered rhesus simian immunodeficiency virus-specific CD8 + T cells reduces the number of transmitted/founder viruses established in rhesus macaques
袋猴工程化感染性病毒特异性 CD8+T 细胞的移植减少了袋猴中建立的传播/创始病毒数量J Virol, 90 (2016), pp. 9942-9952, 10.1128/jvi.01522-16
J Virol，90（2016），第 9942-9952 页，10.1128/jvi.01522-16 - [19]Retroviral transduction and engraftment ability of primate hematopoietic progenitor and stem cells transduced under serum-free versus serum-containing conditions
灵长类造血祖细胞和干细胞在无血清和含血清条件下逆转录引入及植入能力Mol Ther, 5 (2002), pp. 316-322, 10.1006/mthe.2002.0544
Mol Ther，5（2002），pp. 316-322，10.1006/mthe.2002.0544 - [20]Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts
从成年恒河猴成纤维细胞中诱导生成多能干细胞Cell Stem Cell, 3 (2008), pp. 587-590, 10.1016/j.stem.2008.10.014
干细胞，3（2008 年），pp. 587-590，10.1016/j.stem.2008.10.014 - [21]Retroviral transcriptional regulation and embryonic stem cells: war and peace
逆转录调节和胚胎干细胞：战争与和平Mol Cell Biol, 35 (2015), pp. 770-777, 10.1128/mcb.01293-14
Mol Cell Biol，第 35 卷（2015 年），第 770-777 页，DOI：10.1128/mcb.01293-14 - [22]Silencing and variegation of gammaretrovirus and lentivirus vectors
沉默和多色性的 gammaretrovirus 和 lentivirus 载体Hum Gene Ther, 16 (2005), pp. 1241-1246, 10.1089/hum.2005.16.1241
人类基因治疗，16（2005），页 1241-1246，10.1089/hum.2005.16.1241 - [23]PiggyBac transposon-mediated gene transfer in human cells
PiggyBac 转座子介导的基因转移在人类细胞中Mol Ther, 15 (2007), pp. 139-145, 10.1038/sj.mt.6300028
Mol Ther，15（2007 年） ，第 139-145 页，10.1038/sj.mt.6300028 - [24]A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines
一种可选择的全合一 CRISPR 主编辑 piggyBac 转座子可在人类细胞系中实现高效基因编辑Sci Rep, 11 (2021), pp. 1-14, 10.1038/s41598-021-01689-2
Sci Rep，11（2021），pp. 1-14，10.1038 / s41598-021-01689-2 - [25]A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation
基于 piggyBac 的基因组编辑平台和克隆恒河猕猴 iPSC 系列衍生Sci Rep, 11 (2021), pp. 1-17, 10.1038/s41598-021-94419-7
《科学报告》，11 (2021)，页码 1-17，DOI: 10.1038/s41598-021-94419-7 - [26]GFP fluorescence peak fraction analysis based nanothermometer for the assessment of exothermal mitochondria activity in live cells
基于 GFP 荧光峰分析的纳米热计，用于评估活细胞中线粒体的放热活动Sci Rep, 9 (2019), pp. 1-11, 10.1038/s41598-019-44023-7
Sci Rep，9 (2019)，pp. 1-11，10.1038/s41598-019-44023-7 - [27]A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells
用于选择、体内追踪和消融工程细胞的跨基因编码的细胞表面多肽Blood, 118 (2011), pp. 1255-1263, 10.1182/blood-2011-02-337360
血液, 118 (2011), 页码 1255-1263, DOI 10.1182/blood-2011-02-337360 - [28]Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells
用于确定转染细胞中整合 Lentivirus 载体拷贝数的准确验证和优化测试的参考标准Sci Rep, 11 (2021), pp. 1-9, 10.1038/s41598-020-79698-w
Sci Rep，11（2021 年），页码 1-9，DOI 10.1038/s41598-020-79698-w - [29]Integration mapping of piggyBac-mediated CD19 chimeric antigen receptor T cells analyzed by novel tagmentation-assisted PCR
通过新颖的标记辅助 PCR 分析 piggyBac 介导的 CD19 嵌合抗原受体 T 细胞的整合映射EBioMedicine, 34 (2018), pp. 18-26, 10.1016/j.ebiom.2018.07.008
EBioMedicine，34（2018），第 18-26 页，10.1016/j.ebiom.2018.07.008 - [30]Of mice and not men: differences between mouse and human immunology
鼠与人：小鼠和人类免疫学之间的差异J Immunol, 172 (2003), pp. 2731-2738
J Immunol，172（2003），第 2731-2738 页. - [31]Translational impact of NIH-funded nonhuman primate research in transplantation
NIH 资助的非人灵长类转移研究的转化影响Sci Transl Med, 11 (2019), pp. 1-9, 10.1126/scitranslmed.aau0143 - [32]Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus
从非人灵长类诱导多能干细胞编辑的 CCR5 位点生成 SIV 抗性 T 细胞和巨噬细胞Stem Cell Reports, 17 (2022), pp. 953-963, 10.1016/j.stemcr.2022.03.003
多能干细胞报告, 第 17 卷 (2022), 页码 953-963, DOI: 10.1016/j.stemcr.2022.03.003 - [33]Efficient transduction of human and rhesus macaque primary T cells by a modified human immunodeficiency virus type 1-based lentiviral vector
通过改良的人类免疫缺陷病毒类型 1 基于慢病毒载体有效转导人类和恒河猕猴原代 T 细胞Hum Gene Ther, 28 (2017), pp. 271-285, 10.1089/hum.2016.135
人类基因治疗，28（2017 年），271-285 页，10.1089/hum.2016.135 - [34]Aberrant clonal hematopoiesis following lentiviral vector transduction of HSPCs in a rhesus macaque
恒河猕猴 HSPCs 植入后慢病毒载体转导异常的克隆性造血过程Mol Ther, 27 (2019), pp. 1074-1086, 10.1016/j.ymthe.2019.04.003 - [35]Chimeric antigen receptor T cell–mediated neurotoxicity in nonhuman primates
猴嗷叫抗原受体 T 细胞在灵长类动物中引起的神经毒性Cancer Discov, 8 (2018), pp. 750-763, 10.1158/2159-8290.CD-17-1368 - [36]Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells
注射了 piggyBac 修改 CD19 嵌合抗原受体 T 细胞后，产生的产品源性淋巴瘤的研究Blood, 138 (2021), pp. 1391-1405, 10.1182/blood.2021010858
《血液》，138 (2021)，第 1391-1405 页，DOI 号：10.1182/blood.2021010858 - [37]Development of CAR T-cell lymphoma in 2 of 10 patients effectively treated with piggyBac-modified CD19 CAR T cells
在 10 名有效治疗的患者中，有 2 名患者发展为 CAR T 细胞淋巴瘤，这些患者有效治疗了 piggyBac 修饰的 CD19 CAR T 细胞Blood, 138 (2021), pp. 1504-1509, 10.1182/blood.2021010813
血液，138（2021），pp. 1504-1509，10.1182/blood.2021010813 - [38]Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia
靶向抗体介导的小鼠 CD19 CAR T 细胞去除永久逆转 B 细胞再生障碍J Clin Invest, 126 (2016), pp. 4262-4272, 10.1172/JCI84813
J Clin Invest，126（2016），pp. 4262-4272，10.1172/JCI84813 - [39]Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques
缅甸恒河猕猴体内针对类人免疫缺陷病毒感染的强效 CD8+T 细胞的 T 细胞受体的鉴定Biochem Biophys Res Commun, 521 (2020), pp. 894-899, 10.1016/j.bbrc.2019.10.196
生物化学与生物物理学通讯, 521 (2020), 894-899 页, doi: 10.1016/j.bbrc.2019.10.196
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