Use of skin grafts programmed to express VEGF-C with biosensor feedback regulation to treat lymphedema

项目摘要 淋巴水肿是一种病态的肌肉骨骼和皮肤疾病，影响广泛的患者群体。在 在美国，这种情况主要影响那些因癌症接受淋巴结手术的人。 管理;全世界有超过3亿人受到淋巴感染的影响。患肢 变得肿胀，并出现皮肤增厚和纤维脂肪组织沉积。患者会感到疼痛， 肢体功能受损，慢性伤口，并有继发性肿瘤（淋巴管肉瘤）的风险。 不幸的是，目前治疗水肿的治疗策略取得了有限的成功。非手术 包括压迫包裹、手动淋巴引流和气动泵在内的管理需要严格的 对患者来说，这是一个非常耗时的过程。手术方法已证明有效，但需要 专业手术设备和技术培训，限制了向500多万人提供这种选择的能力。 在美国受影响的人。血管内皮生长因子-C（VEGF-C）是一种已知的促血管生成因子， 淋巴管生成生长因子，已被研究作为一种基于药物的方法来治疗继发性 水肿递送策略如腺病毒载体、支架和重复注射已经提供了 VEGF-C的重要概念验证支持。然而，临床翻译尚未实现。 在过去的几年里，我们的实验室开发了一种新的技术，使表皮干细胞 进行基因编辑以表达感兴趣的生物制品。这些干细胞在体外培养以产生皮肤 可以应用于患者的移植物。我们的方法已经证明了治疗糖尿病的有效性， 在小鼠模型中通过全身生物利用度研究可卡因滥用。在这份提案中，我们将建立在我们以前的 研究结果使这项技术更接近临床实用，同时扩大其应用。 首先，我们将编程表皮干细胞表达VEGF-C，并在体外培养这些干细胞， 皮肤移植为了使我们更接近临床转化，表皮干细胞将从人类皮肤中分离出来。我们 将证明皮肤移植物可以产生局部生物利用度的生长因子，从而扩大使用范围。 of this technology技术.我们将在后肢水肿模型中使用这些皮肤移植物，从而证明 该技术具有治疗单侧肢体孤立性水肿的临床应用潜力。 其次，我们将通过引入生物传感器机制来推进当前的皮肤移植技术， 提供VEGF-C表达的负反馈调节。这种生物传感器将积极影响 本发明的目的是提供这种疗法的翻译潜力，并且对于体内“生物反应器”移植物的翻译具有更广泛的实用性。 本研究结束后，我们将解决癌症患者经历的具有挑战性的发病率问题 （继发性淋巴水肿），使用一种新技术（编程表皮干细胞）来扩大其效用， 包括生长因子（VEGF-C）和局部递送。

PROJECT SUMMARY Lymphedema is a morbid musculoskeletal and skin condition affecting a broad population of patients. In the United States, this condition primarily affects individuals who have had lymph node surgery for cancer management; worldwide over 300 million individuals are affected due to lymphatic infection. The affected limb becomes swollen, and develops skin thickening and fibroadipose tissue deposition. Patients experience pain, impaired limb function, chronic wounds, and are at risk for secondary tumors (lymphangiosarcoma). Unfortunately, current therapeutic strategies to treat lymphedema have had limited success. Non-operative management including compression wraps, manual lymphatic drainage, and pneumatic pumps require strict adherence and are time-consuming for patients. Surgical approaches have demonstrated efficacy but require specialized surgical equipment and technical training, limiting the ability to provide this option to the 5+ million affected individuals in the United States. Vascular endothelial growth factor-C (VEGF-C) is a known pro- lymphangiogenic growth factor which has been studied as a drug-based approach to treat secondary lymphedema. Delivery strategies such as adenoviral vectors, scaffolds, and repeat injection have provided important proof-of-concept support for VEGF-C. However, clinical translation has not been realized. Over the past several years, our laboratory has developed a novel technology enabling epidermal stem cells to undergo gene-editing to express biologics of interest. These stem cells are cultured in vitro to produce skin grafts which can be applied to the patient. Our approach has demonstrated efficacy treating diabetes and cocaine abuse through systemic bioavailability in mouse models. In this proposal, we will build on our previous findings to bring this technology closer to clinical utility while broadening its application. First, we will program epidermal stem cells to express VEGF-C and culture these stem cells in vitro, to form skin grafts. To bring us closer to clinical translation, epidermal stem cells will be isolated from human skin. We will demonstrate that skin grafts can produce growth factors for local bioavailability, thereby expanding the use of this technology. We will use these skin grafts in a model of hindlimb lymphedema, thereby demonstrating that this technology has the potential for clinical utility to treat lymphedema isolated to a unilateral limb. Second, we will advance the current skin graft technology by introducing a biosensor mechanism which provides negative feedback regulation of VEGF-C expression. This biosensor will positively impact the translational potential of this therapy, and have broader utility for translation of in vivo “bioreactor” grafts. Upon conclusion of this study, we will have addressed a challenging morbidity experienced by cancer patients (secondary lymphedema), using a novel technology (programmed epidermal stem cells) to expand its utility to include growth factors (VEGF-C) and local delivery.