Isolation of narrow subpopulations of cells using molecular computing cascades

描述(由申请人提供)：我们在这里描述一组计算细胞表面状态的分子。这些组可以被编程为在显示基于抗体的靶向部分的细胞表面上自组织，从而导致在任意狭窄的细胞亚群的表面上显示独特的标签(标签)。决定是显示还是显示 标签不会依赖于存在的抗体，即依赖于多个细胞表面标记，这些标记通过它们的存在或不存在来唯一地定义靶细胞。所得到的标签可用于在存在其他亚群的情况下在单个步骤中分离靶细胞。这种基于多个表面标记的一步分离细胞方法由于其可伸缩性、有效性和温和，将比现有方法具有独特的优势。我们将在一个具有治疗意义的淋巴细胞亚群的例子上研究新技术的范围和局限性，将我们的技术与现有方法和多步骤方案进行比较：目标1：其中分子将计算细胞表面上多个标记的存在。我们将首先评估最多三种CD4CD25和CD25CD154T细胞标志物。然后，我们将演示基于标记的强度分离细胞组的能力，分离NK细胞的两个部分(CD56dim和CD56High)，开发具有阈值(截止值)功能的分子集。目标2：其中分子将评估标记的存在和不存在。在对模型系统进行初步优化后，我们将展示分离常规记忆B细胞(CD19 CD21 CD27 IGD-)和最近表征的非常规记忆B细胞(CD19 CD21-/lowCD27-IGD-CD95)的能力。我们的试剂可以与用于靶向细胞表面标记的任何部分结合，只需进行最小的优化。因此，通过我们的结果，我们将向生物医学社区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体。该方法将在基础科学研究、个性化医学和生物医学工程中有广泛的应用。 公共卫生相关性：我们解决的问题经常出现在生命科学、使用细胞的新兴治疗方法和生物医学工程中：我们需要从细胞的海洋中分离出一个狭窄的亚群，该亚群通过存在和不存在多个细胞表面标记而与所有其他亚群唯一地区分开来。在这项建议中，我们描述了一种温和的制备分离技术的开发和优化，该技术在最少的步骤中产生可行的任意狭窄的细胞亚群。这项技术是基于一组分子根据通过与细胞表面的相互作用实现的一组规则进行自组织，并仅标记我们的目标细胞 与世隔绝。该方法将广泛应用于疾病(如自身免疫和癌症)的发病机制研究、细胞治疗以及组织和器官工程，具有现有细胞分离方法的独特优势。

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. PUBLIC HEALTH RELEVANCE: The problem we address appears often in life sciences, emerging therapeutic methods using cells, and biomedical engineering: from a sea of cells we need to isolate a narrow subpopulation that is uniquely distinguished from all other subpopulations through the presence and absence of multiple cell-surface markers. We describe in this proposal the development and optimization of a mild preparative isolation technique yielding viable arbitrarily narrow subpopulations of cells in a minimal number of steps. The technique is based on sets of molecules self-organizing according to sets of rules implemented through interactions with the cell surfaces, and labeling only cells that we target for isolation. The procedure will be widely applicable in studying pathogenesis of diseases (e.g., autoimmunity and cancer), for cellular therapies, and tissue and organ engineering, offering unique advantages over the existing methods for isolation of cells.