Project #2 - Banchereau

项目2 SLE发病率高，治疗选择有限。约30%的患者对标准治疗无效 (SOC)治疗，包括羟氯喹（HCQ）、糖皮质激素（GC）和吗替麦考酚酯 （MMF）。另外约30%的患者最初反应为活动指数降低（SLEDAI）， 浆母细胞（PB），但随后变得难以治疗。其背后的生物机制 这种对治疗缺乏反应的现象人们知之甚少。因此，为了改善临床 SLE的管理，特别是在目前没有有效治疗选择的约60%的SLE患者中 从长远来看，项目2的目标是描述SOC治疗耐药的机制，并确定 治疗干预的新靶点。在上一个CORT融资周期中，我们专注于描述 来自一位SLE患儿的外周血单个核细胞（PBMC）的转录组 使用单细胞（sc）RNAseq和长读段（LR）测序技术对队列进行测序。我们发现多个细胞 类型-单核细胞，树突状细胞，浆细胞（PC），以及CD 4+和CD 8 + T细胞的亚群-表现出 干扰素（IFN）信号。LRseq发现了SLE特有的>250种同种型，其中包括几种细胞表面 具有免疫调节功能的分子。来自纵向队列的11名患者的初步数据显示， 诊断时SLEDAI高与PB数量升高相关。重要的是， 治疗前样本表明，无应答（NR）患者的PB与应答患者的PB不同 (R)患者，包括R-PB中IFN刺激基因（ISG）的表达增加。最后，我们使用 整体和单核（sn）ATAC-seq以鉴定SLE的表观基因组特征。在这里，我们将联合收割机单个- 功能基因组学扩展纵向队列中的细胞方法，以揭示治疗耐药性 系统性红斑狼疮和新的治疗靶点的途径。在目标1中，我们将确定SOC抗性途径， 使用从30名SLE患者中纵向收集的血液样本，在发病时确定细胞转录水平 并随访至少3年。我们将揭示耐药性的转录特征 通过进行Total-seq并鉴定与疾病严重程度和治疗反应相关的亚型。在 目的2，我们将揭示与SLE和治疗相关的表观基因组特征和调控元件 使用单核ATACseq的抗性，并将这些与目标1中鉴定的转录程序联系起来。 纵向样本对于确定疾病特异性基因组签名是否随时间变化是必不可少的。 治疗期间和/或发作期间。在目标3中，我们将描述耐药PB的表型，并应用于 CRISPR-Cas9功能基因组学方法来验证赋予对大肠杆菌抗性的候选分子 疗法项目2的成功完成将确定治疗的转录和表观基因组签名 SLE PBMC中的耐药性，辅以PB中的重点功能研究，PB是SLE中的关键细胞群， 提供潜在的新的治疗靶点，特别是对SOC耐药的个体。

PROJECT SUMMARY PROJECT 2 SLE has high morbidity and limited treatment options. ~30% of patients fail to respond to the standard-of-care (SOC) treatment, composed of hydroxychloroquine (HCQ), Glucocorticoids (GCs), and mycophenolate mofetil (MMF). Another ~30% of patients initially respond with a decreased activity index (SLEDAI) and a decrease in plasmablasts (PBs), but subsequently become refractory to treatment. The biological mechanisms that underlie this lack of response to treatment are poorly understood. Thus, to progress towards improved clinical management of SLE especially in the ~60% of SLE patients who currently do not have effective treatment options in the long term, the goal of Project 2 is to delineate the mechanisms of resistance to SOC therapy and to identify novel targets for therapeutic intervention. In the previous CORT funding cycle, we focused on characterizing the transcriptomes of peripheral blood mononuclear cells (PBMCs) from a cross-sectional pediatric SLE patient cohort using single-cell (sc) RNAseq and long-read (LR) sequencing technologies. We found that multiple cell types -- monocytes, dendritic cells, plasma cells (PCs), and subsets of CD4+ and CD8+ T cells -- exhibit an interferon (IFN) signature. LRseq uncovered >250 isoforms unique to SLE, which include several cell surface molecules with immune regulatory functions. Preliminary data from 11 patients from a longitudinal cohort showed that a high SLEDAI at diagnosis correlates with elevated numbers of PBs. Importantly, transcriptional profiling of pre-therapy samples suggested that the PBs of non-responder (NR) patients are distinct from those of responder (R) patients, including increased expression of IFN-stimulated genes (ISGs) in R-PBs. Finally, we have used bulk and single-nucleus (sn) ATAC-seq to identify epigenomic signatures of SLE. Here, we will combine single- cell approaches in an expanded longitudinal cohort with functional genomics to uncover treatment resistance pathways in SLE and novel therapeutic targets. In Aim 1, we will identify SOC resistance pathways at the single- cell transcriptional level using blood samples collected longitudinally from 30 SLE patients identified at the onset of disease and followed over at least 3 years. We will uncover transcriptional signatures of therapy resistance by performing Total-seq and identify isoforms associated with disease severity and response to treatment. In Aim 2, we will uncover epigenomic signatures and regulatory elements associated with SLE and therapy resistance using single-nucleus ATACseq and link these to transcriptional programs identified in Aim 1. Longitudinal samples are essential for determining whether disease-specific genomic signatures change upon treatment and/or during flares. In Aim 3, we will characterize the phenotype of therapy-resistant PBs and apply CRISPR-Cas9 functional genomics approaches to validate candidate molecules that confer resistance to therapy. Successful completion of Project 2 will identify transcriptional and epigenomic signatures of therapy resistance in SLE PBMCs, complemented by focused functional studies in PBs, a cell population critical in SLE, to deliver potential novel therapeutic targets especially for individuals resistant to SOC.