Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.
Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.
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DOI：
10.1016/j.jcyt.2023.02.010
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发表时间：
2023-06
期刊：
影响因子：
4.5
通讯作者：
Jin, Ping
中科院分区：
文献类型：
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作者：
Ma, Jinxia;Shao, Lipei;Fuksenko, Tatyana;Liu, Hui;Shi, Rongye;Dinh, Anh;Highfill, Steven L.;Zhang, Nan;Panch, Sandhya R.;Somerville, Robert P.;Stroncek, David F.;Jin, Ping
参考基因是液滴数字聚合酶链式反应 (ddPCR) 等临床检测的重要组成部分，可测量整合到基因工程细胞中的载体拷贝数以及临床细胞疗法中使用的重编程细胞中质粒的丢失。应谨慎选择参考基因，因为已发现不同个体之间基因组片段的拷贝数可能存在数千种变异。此外，在患有癌症和其他增殖性疾病的同一个人中，患病人和健康人的细胞之间基因组的大部分拷贝数也可能不同。本研究的目的是鉴定可用于转导嵌合抗原受体 T 细胞的拷贝数变异分析以及使用 ddPCR 在诱导多能干细胞中进行质粒丢失分析的参考基因。我们使用癌症基因组图谱 (TCGA) 来评估候选参考基因。如果 TCGA 发现某个候选基因在癌症中具有低拷贝数变异，则使用 ddPCR 测量来自健康受试者、癌细胞系以及急性淋巴细胞白血病、淋巴瘤、多发性骨髓瘤和人乳头瘤病毒相关癌症患者的细胞中潜在参考基因的拷贝数。除了我们在拷贝数测定中使用的 rPP30 基因之外，还使用 TCGA 评估了其他三个候选参考基因，该分析发现，在我们机构目前正在接受细胞疗法治疗的所有癌细胞类型中，四个基因区域（AGO1、AP3B1、MKL2 和 rPP30）都没有被扩增或删除。通过 ddPCR 测量的健康受试者细胞中 AP3B1、AGO1、rPP30 和 MKL2 基因的拷贝数相似。我们发现，在一些临床样本中，AGO1 存在拷贝数改变，并且通过 ddPCR 测量的基因 AP3B1、MKL2 和 rPP30 的拷贝数在我们机构目前正在接受基因工程 T 细胞疗法治疗的癌细胞类型患者的细胞中相似。根据我们目前的结果，AP3B1、MKL2和rPP30这三个基因适合用作参考基因，用于测量由急性白血病、淋巴瘤、多发性骨髓瘤和人乳头瘤病毒相关癌症患者产生的嵌合抗原受体T细胞中的载体拷贝数。我们将继续在未来的样品上评估 AGO1。
Reference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people. The purpose of this study was to identify reference genes that could be used for copy number variation analysis of transduced chimeric antigen receptor T cells and for plasmid loss analysis in induced pluripotent stem cells using ddPCR. We used The Cancer Genome Atlas (TCGA) to evaluate candidate reference genes. If TCGA found a candidate gene to have low copy number variance in cancer, ddPCR was used to measure the copy numbers of the potential reference gene in cells from healthy subjects, cancer cell lines and patients with acute lymphocytic leukemia, lymphoma, multiple myeloma and human papillomavirus–associated cancers. In addition to the rPP30 gene, which we have has been using in our copy number assays, three other candidate reference genes were evaluated using TCGA, and this analysis found that none of the four gene regions (AGO1, AP3B1, MKL2 and rPP30) were amplified or deleted in all of the cancer cell types that are currently being treated with cellular therapies by our facility. The number of copies of the genes AP3B1, AGO1, rPP30 and MKL2 measured by ddPCR was similar among cells from healthy subjects. We found that AGO1 had copy number alteration in some of the clinical samples, and the number of copies of the genes AP3B1, MKL2 and rPP30 measured by ddPCR was similar among cells from patients with the cancer cell types that are currently being treated with genetically engineered T-cell therapies by our facility. Based on our current results, the three genes, AP3B1, MKL2 and rPP30, are suitable for use as reference genes for assays measuring vector copy number in chimeric antigen receptor T cells produced from patients with acute leukemia, lymphoma, multiple myeloma and human papillomavirus–associated cancers. We will continue to evaluate AGO1 on our future samples.
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DOI：
10.1097/cji.0b013e318194a6e8
发表时间：
2009-02
期刊：
Journal of immunotherapy (Hagerstown, Md. : 1997)
影响因子：
--
作者：
Hollyman D;Stefanski J;Przybylowski M;Bartido S;Borquez-Ojeda O;Taylor C;Yeh R;Capacio V;Olszewska M;Hosey J;Sadelain M;Brentjens RJ;Rivière I
通讯作者：
Rivière I
影响因子：
12.3
作者：
Stodolna A;He M;Vasipalli M;Kingsbury Z;Becq J;Stockton JD;Dilworth MP;James J;Sillo T;Blakeway D;Ward ST;Ismail T;Ross MT;Beggs AD
通讯作者：
Beggs AD
影响因子：
20.3
作者：
Kochenderfer, James N.;Wilson, Wyndham H.;Rosenberg, Steven A.
通讯作者：
Rosenberg, Steven A.
影响因子：
2.7
作者：
Naumann, S;Reutzel, D;Decker, HJ
通讯作者：
Decker, HJ
影响因子：
8.8
作者：
通讯作者：
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