Novel Highly Regenerative and Scalable Progenitor Cell Exosomes for Treating Peripheral Artery Disease

摘要 外周动脉疾病(PAD)与四肢血管阻塞有关，导致间歇性 跛行、缺血性溃疡、休息时腿痛和截肢。在美国，大约有650万人 外周动脉疾病(PAD)， 影响到12-20%的60岁以上的人 。目前的细胞疗法 由于移植细胞存活率低和不足，恢复肢体灌流仅显示出适度的益处 有利于血管生成的线索。因此，迫切需要PAD疗法的替代疗法。最近， 使用间充质干细胞(MSCs)来源的外切体治疗已经被认为是一种有前途的无细胞治疗方法。 治疗各种疾病，包括PAD。然而，MSCs是异质的，不能很好地扩展，因此， 限制了它们作为商业规模开发和临床的可靠外切体来源的有效性 申请。我们在AgeX治疗公司之前已经证明，我们高度再生的、专有的 处于早期发育状态的PureStem®祖细胞能产生高度血管生成外切体。 因此，我们建议使用AgeX的高度可伸缩性来克服当前Exosome生产的限制 PureStem®人类多能干细胞来源细胞系作为同种异体PAD疗法的来源。一个关键的理由 因为使用外切体是因为它们的免疫原性很低，这意味着它们不太可能引起不良反应。 免疫反应，而且它们是非复制性的，这避免了与细胞治疗相关的许多风险。 由于外切体介导胞间通讯，我们可以使用PureStem而不是管理细胞 血管生成外切体在PAD小鼠模型中改善肢体血运重建和血液灌流。此外， 外切体的生产可能更具成本效益，使用和储存也更方便。 这一STTRI期提案的目标是证明外切体的治疗可行性，以改善 小鼠后肢缺血模型的血管生成功能。首先，我们将建立两个先导血管生成外切体 生产细胞系并在体外生产与GMP相容的PureStem®外切体 外切体的产量、外切体的血管生成活性以及外切体的可扩展性和稳定性。然后我们将测试这两个 在小鼠后肢缺血模型中筛选PureStem外切体系以验证其对小鼠的治疗效果 活着。在我们已建立的PAD动物模型中使用可伸缩的Exosome的体内可行性的证据 PureStem细胞系将为进一步的临床前开发和IND铺平道路，从而使 总而言之，我们预计PureStem外切体的治疗潜力 提供一种创新、经济、方便的“现成”再生干细胞衍生疗法 垫子。

ABSTRACT Peripheral artery disease (PAD) is associated with vascular obstructions in the extremities, leading to intermittent claudication, ischemic ulcers, leg pain at rest, and amputation. In the US, approximately 6.5 million people have peripheral artery disease (PAD), affecting 12-20% of people over 60 years of age . Current cell therapies to restore limb perfusion have shown only moderate benefits due to poor transplant cell survival and inadequate pro-angiogenic cues. Therefore, there is a critical need for alternative treatments for PAD therapy. Recently, therapy using mesenchymal stem cell (MSCs)-derived exosomes has been recognized as a promising, cell-free treatment in various diseases, including PAD. However, MSCs are heterogeneous and do not scale well, thus, limiting their usefulness as a reliable source of exosomes for commercial scale development and clinical application. We at AgeX Therapeutics have previously shown that our highly regenerative, proprietary PureStem® progenitor cells, which are at an early developmental state, produce highly angiogenic exosomes. Thus, we propose to overcome the current exosome production limitations by using AgeX’s highly scalable PureStem® human pluripotent stem cell-derived cell lines as a source of allogeneic PAD therapy. A key rationale for using exosomes is that they have low immunogenicity, which means they are not likely to cause undesirable immune responses, and they are non-replicative, which obviates many of the risks associated with cell therapy. Since exosomes mediate intercellular communication, instead of administering cells, we can use PureStem angiogenic exosomes in a murine model of PAD to improve limb revascularization and blood perfusion. Moreover, exosomes are likely to be more cost-effective to produce and more convenient to use and store. The goal of this STTR Phase I proposal is to demonstrate the therapeutic feasibility of exosomes to improve angiogenic function in a mouse hindlimb ischemia model. First, we will establish two lead angiogenic exosome production cell lines and produce GMP-compatible PureStem® exosomes in vitro by assessing lines based on exosome output, exosome angiogenic activity, and exosome scalability and stability. We will then test the two selected PureStem exosome lines in a hindlimb ischemia mouse model to validate their therapeutic efficacy in vivo. Evidence of in vivo feasibility in our well-established PAD animal model using exosomes from a scalable PureStem cell line will pave the way for further preclinical development and IND, thus enabling studies for a prospective STTR Phase II. Taken together, we anticipate the therapeutic potential for PureStem exosomes to provide an innovative, cost-effective, and convenient “off-the-shelf” regenerative stem cell-derived therapy for PAD.