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

器官移植通常用于替换具有各种健康问题的患者的整个器官，但由于缺乏供体，目前有超过10万名患者等待接受移植，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。在这项提案中，我们的目标是开发一个简单的平台，在此平台上，人体器官可以在猪体内生长，并通过产生猪-猪嵌合体来证明这一点的可行性，在猪-猪嵌合体中，胰腺及其相关的血管系统完全来自供体猪细胞。我们将使用胚泡互补，这已被证明在小鼠和猪，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ES细胞的可用性、成本和可行性，并允许我们查询用于猪外源器官产生的许多发育基因。第二，我们将采用Prophetics的创新和专有的牲畜基因编辑技术，快速轻松地消除多个基因的功能，从而生产出具有多个供体来源细胞谱系的嵌合动物。具体地说，将敲除形成胰腺和胰腺血管所必需的两个基因，从而允许在含有全部仅来源于供体的血管的猪中形成外源性胰腺。该平台将加速外源器官生产领域的发展，并满足NIH研讨会上确定的几个需求，以改善再生医学的动物模型。在后续的II期研究中，我们将评估与人多能细胞互补后多谱系消融宿主的器官发生。

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.