Targeting Therapeutic Molecules to Dystrophic Muscle via the Immune System

描述（由申请人提供）：治疗杜氏肌营养不良症的药理学方法的主要挑战是在正确的时间将正确的分子靶向正确的位置。即使当治疗性分子的全身递送对患病组织具有有益作用时，脱靶效应也可产生负面后果，其减损特定治疗剂的临床使用。我们提出了一种创新的策略，利用人体自身的能力，在病变最明显的部位和时间将分子输送到病变组织。我们将使用表达治疗分子（LIF）转基因的骨髓细胞（BMC），该转基因由在活化的巨噬细胞中高度表达的启动子（CD11b）驱动，并将这些细胞移植到患有肌营养不良症的小鼠中。由于mdx小鼠肌营养不良的发病和严重程度反映了表达高水平CD11b的髓样细胞对肌肉的侵袭，我们预计治疗性LIF的递送将跟踪疾病的发生和严重程度。因此，我们希望实现“按需”将治疗分子特异性靶向递送至患病组织，以最小化负面的脱靶效应。如果成功的话，我们的发现可以为治疗肌肉萎缩症提供一个全新的途径。在我们提出的研究中，我们将探索使用骨髓来源的髓样细胞作为载体，在两个目标中向营养不良的肌肉递送治疗性货物。目标1.转基因骨髓基质细胞移植能否提供一种机制，将治疗分子靶向营养不良的肌肉？我们将从三个转基因供体系中的每一个移植BMC，这些供体系显示LIF转基因表达在分化的白细胞中的野生型水平的8至131倍。将评估mdx小鼠的病理学减少、巨噬细胞从溶细胞表型转变为促再生表型以及肌肉功能改善。我们还将分析是否发生负面脱靶效应。目标2.受体小鼠的非清髓性预处理是否允许足够的转基因供体髓系细胞植入，以提供病理学拯救，而没有负面的脱靶效应？在目标2中，我们将测试在骨髓移植前使用药理学方法进行非清髓性预处理是否会产生LIF转基因对肌肉的充分靶向和肌肉病理的减弱，而不会引起负面的脱靶效应。

DESCRIPTION (provided by applicant): A major challenge for pharmacological approaches to treating Duchenne muscular dystrophy is targeting the right molecule to the right place at the right time. Even when systemic delivery of a therapeutic molecule has beneficial effects on diseased tissue, off-target effects can produce negative consequences that detract from the clinical use of a particular therapeutic agent. We propose an innovative strategy to exploit the body's own ability to deliver molecules to diseased tissue at sites and times when pathology is most pronounced. We will use bone marrow cells (BMCs) that express a transgene for a therapeutic molecule (LIF) that is driven by a promoter (CD11b) that is highly expressed in activated macrophages and transplant those cells into mice that have muscular dystrophy. Because the onset and severity of muscular dystrophy in mdx mice mirrors the invasion of muscle with myeloid cells that express high levels of CD11b, we anticipate that delivery of therapeutic LIF will track the occurrence and severity of the disease. Thus, we hope to achieve targeted delivery of a therapeutic molecule specifically to diseased tissue "on demand," to minimize negative, off-target effects. If successful, our findings could provide a wholly-new avenue for the treatment of muscular dystrophy. In our proposed investigation, we will explore the use of bone-marrow-derived myeloid cells as vectors for delivery of therapeutic cargo to dystrophic muscle in two aims. Aim 1. Can transplantation of genetically-modified BMCs provide a mechanism to target therapeutic molecules to dystrophic muscle? We will transplant BMCs from each of three transgenic, donor lines that display LIF transgene expression that ranges from 8- to 131- fold wild-type levels in differentiated leukocytes. Recipient, mdx mice will be assessed for reductions in pathology, shifts in macrophages from a cytolytic to a pro-regenerative phenotype and improved muscle function. We will also assay for the occurrence of negative, off-target effects. Aim 2. Can non-myeloablative conditioning of recipient mice allow sufficient engraftment of transgenic, donor myeloid cells to provide a rescue from pathology, without negative, off-target effects? In Aim 2, we will test whether the use of pharmacological approaches to non-myeloablative conditioning prior to bone marrow transplantation will yield sufficient targeting of the LIF transgene to muscle and attenuation of muscle pathology without incurring negative, off-target effects.