Develop novel assays for assessing cellular and gene therapies

开发评估细胞和基因疗法的新方法

• 批准号: 10913195
• 负责人: David Stroncek
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10913195
• 关键词: B-Lymphocytes; Biological Assay; Cell Culture Techniques; Cell Therapy; Cell physiology; Cells; Clinical; Clinical effectiveness; Complex; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Profiling; Genes; Genome; Hematologic Neoplasms; Hematopoietic stem cells; Leukocytes; Measures; Messenger RNA; MicroRNAs; Outcome; Production; Site; T-Lymphocyte; Testing; Time; Transfusion; Unmarried person; cellular engineering; chimeric antigen receptor; chimeric antigen receptor T cells; enzyme linked immunospot assay; gene therapy; improved; metabolomics; miRNA expression profiling; monocyte; next generation sequencing; novel; peripheral blood; single cell analysis; sterility testing; transcriptome; vector

Cell and gene therapy products must be tested for sterility, stability, purity and potency. In addition, it is important to test clinical cell therapy products for identity, consistency and comparability. Testing cellular and gene therapies is challenging. These therapies are generally collected from a single person so the quantity of material available to test is limited. They are typically transfused immediately or shortly after they are produced so there is a very limited amount of time to complete the assays. Many of these therapies are complex cells that have multiple functions. The cell functions that are critical to the clinical effectiveness of these therapies are often not known. Traditionally, analytic assays such as flow cytometry, ELISA, ELISPOT and cell culture have been used to analyze cellular and gene therapies. While these assays have proven to be very useful, the number and types of factors that can be analyzed with these assays is limited. We have been investigating the use of gene and micro RNA expression assays for the analysis of cellular therapies. These assays can require the use of only small quantities of cells and can be used to assess the expression of the entire transcriptome. We have been testing the ability of global gene and micro RNA expression profiling to determine the utility of these assays for assessing the stability, purity and potency of cellular therapies. We have shown that gene expression profiling can detect changes in stored cells and detect differences between peripheral blood leukocytes (T cells, B cells and monocytes) and hematopoietic stem cells. Gene expression profiling has also been able to detect differences between immature and mature dendritic cells (DCs) and has been useful for comparing mature DCs produced using different combinations of maturation agents. Chimeric Antigen Receptor (CAR) T cells are being used to treat a number of hematologic malignancies, however, clinical outcomes have varied among recipients of these therapies and some of this variability is likely due to variability, and hence, differences in potency among CAR T cell products. We are using gene expression analysis, mRNA analysis, single cell analysis, next generation sequencing, vector insertion site and metabolomics to identify factors associated with the clinical potency of these cells. We have also developed an assay that measures the number of copies of the CAR vector that have integrated into the genome of each T cell.

细胞和基因治疗产物必须测试不育，稳定性，纯度和效力。此外，重要的是测试临床细胞治疗产品的身份，一致性和可比性。 测试细胞和基因疗法具有挑战性。这些疗法通常是从一个人那里收集的，因此可以测试的材料数量有限。它们通常在生产后立即或不久将其输血，因此完成测定的时间非常有限。这些疗法中的许多是具有多个功能的复杂细胞。对于这些疗法的临床有效性至关重要的细胞功能通常不知道。传统上，流式细胞仪，ELISA，ELISPOT和细胞培养等分析测定已用于分析细胞和基因疗法。尽管事实证明这些测定法非常有用，但可以用这些测定法分析的因素数量和类型是有限的。 我们一直在研究使用基因和微RNA表达测定法对细胞疗法的分析。这些测定方法只能仅使用少量细胞，可用于评估整个转录组的表达。我们一直在测试全局基因和微RNA表达分析的能力，以确定这些测定方法评估细胞疗法的稳定性，纯度和效力。我们已经表明，基因表达分析可以检测存储的细胞的变化，并检测外周血白细胞（T细胞，B细胞和单核细胞）和造血干细胞之间的差异。基因表达分析还能够检测未成熟和成熟的树突状细胞（DC）之间的差异，并且对于使用成熟剂的不同组合产生的成熟DC非常有用。 嵌合抗原受体（CAR）T细胞用于治疗许多血液系统恶性肿瘤，但是，这些疗法的受体中的临床结果有所不同，其中某些可变性可能是由于变异性，因此CAR T细胞产物之间的效力差异。 我们正在使用基因表达分析，mRNA分析，单细胞分析，下一代测序，载体插入位点和代谢组学来识别与这些细胞临床效力相关的因素。我们还开发了一个测定法，该测定法测量已整合到每个T细胞基因组中的CAR载体的副本数量。

项目成果

High efficiency closed-system gene transfer using automated spinoculation.

• DOI: 10.1186/s12967-021-03126-4
• 发表时间: 2021-11-24
• 期刊: Journal of translational medicine
• 影响因子: 7.4
• 作者: Remley VA;Jin J;Sarkar S;Moses L;Prochazkova M;Cai Y;Shao L;Liu H;Fuksenko T;Jin P;Stroncek DF;Highfill SL
• 通讯作者: Highfill SL

Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.

• DOI: 10.1016/j.jcyt.2023.02.010
• 发表时间: 2023-06
• 期刊: CYTOTHERAPY
• 影响因子: 4.5
• 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
• 通讯作者: Jin, Ping

Evaluation of 3 clinical dendritic cell maturation protocols containing lipopolysaccharide and interferon-gamma.

• DOI: 10.1097/cji.0b013e31819e1773
• 发表时间: 2009-05
• 期刊: Journal of immunotherapy (Hagerstown, Md. : 1997)
• 作者: Han TH;Jin P;Ren J;Slezak S;Marincola FM;Stroncek DF
• 通讯作者: Stroncek DF

Interferon-γ and Tumor Necrosis Factor-α Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype.

• DOI: 10.1038/srep26345
• 发表时间: 2016-05-23
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Jin P;Zhao Y;Liu H;Chen J;Ren J;Jin J;Bedognetti D;Liu S;Wang E;Marincola F;Stroncek D
• 通讯作者: Stroncek D

Pilot analysis of cytokines levels in stored granulocyte-colony-stimulating factor-mobilized peripheral blood stem cell concentrates.

对储存的粒细胞集落刺激因子动员的外周血干细胞浓缩物中细胞因子水平的初步分析。

• DOI: 10.1111/j.1537-2995.2010.02695.x
• 发表时间: 2010
• 期刊: Transfusion
• 影响因子: 2.9
• 作者: Woods,Iyonna;Tawab-Amiri,Abdul;Byrne,Karen;Sabatino,Marianna;Stroncek,DavidF
• 通讯作者: Stroncek,DavidF