Engineered neuroprotective stem-cell exosomes for in utero spina bifida therapy

摘要 脊膜脊膜膨出(MMC)是脊柱裂最严重的形式，也是最常见的先天性病因。 在美国，每天大约有四个孩子出生时就有这种毁灭性的疾病 疾病。MMC是由于神经管的不完全闭合和上覆脊椎的缺失造成的 脊髓暴露于宫内机械和化学创伤。这种创伤会导致终身瘫痪， 后脑引起的肠道和膀胱功能障碍、肌肉骨骼畸形和认知障碍 疝气。宫内手术修复可以改善发病率，但功能恢复是不完整的，大多数 孩子们仍然不能独立行走。我们开发了一种治疗MMC的方法，增强了标准 值得注意的是，在子宫内MMC手术修复，胎盘间充质基质/干细胞(PMSCs)。我们发现 在宫内修复期间使用PMSCs治疗可以预防已建立的胚胎绵羊的后肢瘫痪 MMC模型中，由于PMSC分泌旁分泌神经保护因子。然而，我们证实，私营军事和保安公司 没有长期植入和治疗的羔羊出现了严重的后凸畸形，导致脊髓压迫和 由于缺乏骨骼和邻近的脊柱旁肌肉而形成的系链，这与人类MMC是一致的 肌肉骨骼畸形。为了满足对MMC长期治疗的需求，我们探索了使用 生物工程多功能组合支架。Exosome是一种细胞外小泡，它在 在细胞间通讯中的角色。我们证实了PMSCs分泌的外切体(PMSC-exosome)能够 显著的神经保护功能，在大小上类似于从中衍生出的活体PMSCs。在这 研究中，我们建议开发一种工程化水凝胶系统，允许局部和持续释放 PMSC-exosome到脊髓，这反过来将为治疗MMC提供持续的神经保护 出生。此外，我们的目标是通过用一种覆盖缺陷的方法来延长宫内治疗的寿命 以生物材料为基础的骨支架，提供结构和功能支持。我们计划优化 生物支架对胎鼠和兔的神经保护和再生功能 MMC的模型。我们将在我们的金标准中对最终的多功能组合生物支架产品进行评估 MMC胎羊模型的建立。这种疗法将是无细胞的，现成的，并且易于使用。如果成功，这将是 将使用新的方法在出生前治疗MMC，由于其再生能力，治疗将 改善这些患者的生活质量，并显著降低与以下方面相关的医疗成本 目前的治疗方法。

ABSTRACT Myelomeningocele (MMC) is the most severe form of spina bifida (SB) and the most common congenital cause of lifelong paralysis in the United States, where approximately four children are born daily with this devastating disease. MMC results from the incomplete closure of the neural tube and absent overlying spine leaving the spinal cord exposed to intrauterine mechanical and chemical trauma. This trauma results in lifelong paralysis, bowel and bladder dysfunction, musculoskeletal deformities, and cognitive disabilities due to hindbrain herniation. In utero surgical repair improves morbidity, but functional recovery is incomplete and the majority of children are still unable to walk independently. We developed a treatment for MMC that augments the standard of care, in utero MMC surgical repair, with placental mesenchymal stromal/stem cells (PMSCs). We found that treatment with PMSCs, during in utero repair, prevents hind limb paralysis in the well-established fetal ovine model of MMC, due to PMSC paracrine secretion of neuroprotective factors. However, we confirmed that PMSCs did not engraft long-term and treated lambs developed severe kyphosis causing spinal cord compression and tethering due to the lack of bone and adjacent paraspinal muscles, which is consistent with human MMC musculoskeletal deformities. To meet this need for a long-lasting therapy for MMC, we explored the use of bioengineered multifunctional combination scaffolds. Exosomes are extracellular vesicles that play significant roles in cell-to cell communication. We confirmed that exosomes secreted by PMSCs (PMSC-exosomes) exert significant neuroprotective functions, similar in magnitude to the live PMSCs from which they are derived. In this study, we propose to develop an engineered hydrogel system that will allow for both local and sustained release of PMSC-exosomes to the spinal cord, which in turn will provide sustained neuroprotection to treat MMC before birth. In addition, we aim to increase the longevity of the in utero treatment by covering the defect with a biomaterial-based bony scaffold to provide structural and functional support. We plan to optimize the neuroprotective and regenerative functions of the bioscaffold using our well-established fetal rodent and rabbit models of MMC. We will evaluate the final combination multifunctional bioscaffold product in our gold-standard fetal ovine model of MMC. This therapeutic will be cell-free, off-the-shelf, and easy-to-use. If successful, this novel approach will be used to treat MMC before birth, and due to its regenerative qualities, the treatment will improve the quality of life of these patients, as well as significantly lower the healthcare costs associated with the current treatment.