Multiplex gene-editing to create multi-lineage ablated hosts for exogenic organ production

描述（由申请人提供）：器官移植通常用于替换患有各种健康问题的患者的整个器官，但目前有超过10万名患者由于缺乏供体而等待接受移植，并且这一数字正在急剧上升。外源性器官生产有可能缓解可用器官的缺乏，并帮助迫切需要的患者。在这个提议中，我们的目标是开发一个简单的平台，在这个平台上，人类器官可以在猪身上生长，并通过产生猪-猪嵌合体来证明这一点的可行性，在这种嵌合体中，胰腺及其相关的血管系统完全来自供体猪细胞。我们将使用囊胚互补，这已经被证明在小鼠和猪身上都有效，在这里，我们将解决一些主要问题，使这项技术在临床上适用。首先，我们将评估可用于供体细胞的各种多能细胞，以克服使用猪或人胚胎干细胞的可用性、成本和可行性，并使我们能够查询猪外源器官生产的许多发育基因。其次，我们将采用Recombinetics的创新和专有牲畜基因编辑技术，快速轻松地去除多个基因的功能，从而生产具有多种细胞系的嵌合动物。具体来说，胰腺和胰腺血管形成所必需的两个基因将被敲除，从而允许在猪体内形成外源性胰腺，这些胰腺的血管完全来自供体。该平台将加速外源性器官生产领域，并满足美国国立卫生研究院改善再生医学动物模型研讨会确定的几个需求。在后续的II期研究中，我们将评估多系消融宿主与人类多能细胞互补后的器官发生。

DESCRIPTION (provided by applicant): Organ transplantation is routinely used to replace entire organs in patients with a variety of health issues, yet there are currently more than 100,000 patients waiting to receive transplants due to lack of donors, and this number is rising dramatically. Exogenic organ production has the potential to alleviate the lack of available organs and help patients who are in desperate need. In this proposal, we aim to develop a facile platform upon which human organs can be grown in a pig, and demonstrate the feasibility of this by generating pig-pig chimeras in which the pancreas and its associated vasculature are entirely derived from donor pig cells. We will be using blastocyst complementation, which has been demonstrated to work in both mouse and pig, and here, we will address some major issues to make this technique clinically applicable. First, we will assess various pluripotent cells that coud be used for donor cells, to overcome the availability, cost and feasibility of using pig or human ES cells and allow us to query a number of developmental genes for exogenic organ production in the pig. Second, we will employ Recombinetics' innovative and proprietary livestock gene editing techniques to quickly and easily ablate the function of multiple genes, allowing the production of chimeric animals with multiple cell lineages being donor-derived. Specifically, two genes that are necessary for the formation of the pancreas and pancreatic vasculature will be knocked out, allowing for the formation of an exogenic pancreas in pigs harboring vessels that are all solely donor-derived. This platform will accelerate the field of exogenic organ production and meets several needs identified in the NIH Symposium for Improving Animal Models for Regenerative Medicine. In follow-on Phase II studies, we will evaluate organogenesis in multi-lineage ablated hosts after complementation with human pluripotent cells.