Cell specific delivery of novel therapies to enhance glomerular regeneration and repair

项目概要/摘要 该项目的目标是改变蛋白尿性肾小球疾病的治疗模式， 通过细胞特异性递送以增强体内足细胞修复和再生的治疗剂开发。 足细胞，高度特化的终末分化上皮细胞，在大多数的 肾小球疾病由于足细胞不能自我更新，足细胞损失导致肾小球瘢痕形成。一 壁上皮细胞（佩奇）的亚群可以作为足细胞干细胞（PEC祖细胞），但它们的 再生潜力不足以克服疾病相关的肾小球损伤。增强 因此，足细胞的有效修复需要双重同步方法：（i）替换丢失的足细胞， 增加它们的数量，和（ii）限制/逆转对剩余足细胞的损伤。然而，少校 知识差距使我们无法实现这些目标，其中包括我们对分子生物学的有限知识。 刺激PEC自我更新和足细胞再生/修复的因子，以及 在体内将这些因子递送到特定的肾细胞类型。 我们的四名专家调查员团队将利用互补工具来缩小这些知识差距， 产生创新疗法。Wessely博士将应用实验设计（DoE）方法来识别新的 增加PEC祖细胞和减少足细胞损失的分子组合;罗伯茨博士将结合 将这些治疗剂转化为VHH（纳米抗体），用于递送至PEC祖细胞和足细胞; Freedman博士将 生成基因编辑的人类肾脏类器官，以验证VHH与来自 Shankland博士将使用足细胞耗竭和人类类器官的谱系追踪动物模型 用于体内安全性和功效分析。这条管道最终将测试 靶向递送PEC和足细胞特异性治疗性货物可以增强足细胞 在体内修复和再生，并将肾小球功能恢复到临床疾病阈值以下。 这项工作将通过两个具体目标来完成，每个目标都有独特的里程碑。第一个目标是 通过肽和小分子新组合的细胞靶向递送增加体内肾小球再生 分子以增加壁上皮细胞来源的足细胞祖细胞。第二个目标是增加 通过新鉴定的疗法的细胞类型特异性递送来有效修复受损的足细胞。为 目的，我们将采用上述管道，由DoE发现候选疗法，并与 肾小球疾病的特征。这些将与细胞类型特异性VHH组合， 更高多样性的重组VHH文库以选择性地将它们递送至人佩奇（Aim 1）或足细胞（Aim 2）。增强的体内再生将在FSGS的动物模型和移植的人中得到证实。 类器官这一过程将为肾脏疾病的治疗建立一个新的范例， 用于进一步临床前开发和最终人体临床试验的治疗候选物。

PROJECT SUMMARY/ABSTRACT The goal of this project is to change the treatment paradigm for proteinuric glomerular diseases by combining therapeutics development with cell-specific delivery to enhance podocyte repair and regeneration in vivo. Podocytes, highly specialized terminally differentiated epithelial cells, are injured in the majority of glomerular diseases. As podocytes cannot self-renew, podocyte loss leads to glomerular scarring. A subpopulation of parietal epithelial cells (PECs) can serve as podocyte stem cells (`PEC progenitors'), but their regenerative potential is insufficient to overcome disease-associated glomerular damage. Enhancing productive repair of podocytes thus requires a dual synchronized approach: (i) replacing lost podocytes to increase their number, and (ii) limiting/reversing damage to the remaining podocytes. However, major knowledge gaps prevent us from achieving these goals; these include our limited knowledge on the molecular factors stimulating PEC self-renewal and podocyte regeneration/repair, as well as options methods for delivering these factors to specific kidney cell types in vivo. Our team of four expert investigators will wield complementary tools to close these knowledge gaps and produce innovative therapies. Dr. Wessely will apply Design of Experiment (DoE) approaches to identify novel combinations of molecules that increase PEC progenitors and reduce podocyte loss; Dr. Roberts will conjugate these therapeutics to VHHs (nanobodies) for delivery to PEC progenitors and podocytes; Dr. Freedman will generate gene-edited human kidney organoids to validate effects of VHHs compared to clinical data from patients; Dr. Shankland will use lineage tracing animal models of podocyte depletion and human organoids transplanted into mouse kidneys for in vivo safety and efficacy analysis. This pipeline will ultimately test the hypothesis that targeted delivery of PEC- and podocyte-specific therapeutic cargos can enhance podocyte repair and regeneration in vivo, and restore glomerular function to below the clinical disease threshold. The work will be accomplished through two Specific Aims, each with unique Milestones. The first Aim is to increase glomerular regeneration in vivo by cell targeted delivery of novel combinations of peptides and small molecules to augment podocyte progenitors of parietal epithelial cell origin. The second Aim is to increase productive repair of damaged podocytes by cell-type specific delivery of newly identified therapies. For both aims, we will employ the above pipeline to discover candidate therapeutics by DoE and cross-referenced with glomerular disease signatures from human patients. These will be combined with cell type-specific VHHs from high diversity recombinant VHH libraries to selectively deliver them to human PECs (Aim 1), or podocytes (Aim 2). Enhanced regeneration in vivo will be demonstrated in animal models of FSGS and transplanted human organoids. This process will establish a new paradigm for the treatment of kidney disease, and produce lead therapeutic candidates for further pre-clinical development and ultimately human clinical trials.