Isolation of narrow subpopulations of cells using molecular computing cascades

描述（由申请人提供）：我们在此描述了计算细胞表面状态的分子组。这些组可以被编程以在细胞表面上自组织，从而以导致在任意窄的细胞亚群的表面上显示独特标签（标记）的方式显示基于抗体的靶向部分。决定展示或 标签不依赖于存在的抗体，即，在多个细胞表面标记上，通过它们的存在或不存在唯一地限定靶细胞。所得标签可用于在其它亚群存在下在单个步骤中分离靶细胞。这种基于多个表面标志物分离细胞的单步方法由于其可扩展性、有效性和温和性而具有优于现有方法的独特优势。我们将研究新技术的范围和局限性的治疗感兴趣的淋巴细胞亚群的例子，比较我们的技术与现有的方法和多步骤协议：目的1：其中的分子将计算细胞表面上的多个标记的存在。我们将从评估CD 4 + CD 25+和CD 4 + CD 25 + CD 154 + T细胞上的多达三种标志物开始。然后，我们将展示基于标记物的强度分离细胞组的能力，分离NK细胞的两个部分（CD 56 dim和CD 56 high），开发具有阈值（截止值）功能的分子组。目标2：其中分子将评估标记物的存在和不存在。在对模型系统进行初始优化后，我们将证明分离常规记忆B细胞（CD 19 + CD 21 + CD 27 +IgD-）和最近表征的非常规记忆B细胞（CD 19 + CD 21-/低CD 27-IgD-CD 95+）的窄亚群的能力。 我们的试剂可以与任何用于靶向细胞表面标志物的部分组合，仅需最小的优化。因此，通过我们的研究结果，我们将向生物医学界介绍一种高度模块化的方法，使用现成的试剂分离狭窄的细胞群。该方法将在基础科学研究、个性化医疗和生物医学工程中有广泛的应用。

DESCRIPTION (provided by applicant): We describe here sets of molecules that compute the state of the cell surface. The sets can be programmed to self-organize on cell surfaces displaying antibody-based targeting moieties in a way that results in unique tags (labels) displayed on the surfaces of arbitrarily narrow subpopulations of cells. The decision to display or not the tag will depend on antibodies that are present, i.e., on the multiple cell surface markers that define uniquely the targeted cells via their presence or absence. The resulting tag can be used to isolate in a single step the targeted cells in the presence of other subpopulations. This single-step method for isolation of cells based on multiple surface markers will have unique advantages over existing methods because of its scalability, efficacy, and mildness. We will study the scope and limitations of new technique on an example of therapeutically interesting subpopulations of lymphocytes, comparing our technique with existing methods and multistep protocols: Aim 1: in which the molecules will compute the presence of multiple markers on a cell surface. We will start with evaluation of up to three markers on CD4+CD25+ and CD4+CD25+CD154+ T- cells. We will then demonstrate the ability to isolate groups of cells based on the intensity of a marker, separating two fractions of NK cells (CD56dim and CD56high), developing sets of molecules with thresholding (cut off value) function. Aim 2: in which molecules will evaluate both the presence and absence of markers. After initial optimization on a model system, we will demonstrate the ability to isolate narrow subpopulations of conventional memory B-cells (CD19+CD21+CD27+IgD-) and recently characterized unconventional memory B-cells (CD19+CD21-/lowCD27-IgD-CD95+). Our reagents can be combined with any moiety used for targeting cell-surface markers with only minimal optimization. Thus, through our results we will introduce the biomedical community to a highly modular approach to isolating narrow populations of cells using of-the- shelf reagents. The method will have wide applications in basic science research, personalized medicine, and biomedical engineering.