Data_Sheet_1_Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART−/−IL2RG−/Y SCID Pigs.docx

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T− B− NK− SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)−/− genetic background. We confirmed ART−/−IL2RG−/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART−/−IL2RG−/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART−/−IL2RG−/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

重症联合免疫缺陷（SCID）猪是一类新兴的生物医学动物模型。相较于小鼠，猪在解剖学与生理学特征上更接近人类，因此成为临床前再生医学与癌症研究领域极具价值的实验工具。进一步开发该模型的核心步骤之一，是实现SCID猪的免疫人源化。本研究在自然发生的DCLRE1C（ARTEMIS）缺陷遗传背景下，通过对IL2RG基因进行定点CRISPR/Cas9诱变，成功构建了T、B、NK细胞缺失的SCID猪模型。研究证实，ART缺陷且IL2RG缺陷的雄性（ART−/−IL2RG−/Y）猪的外周血与淋巴组织中均未检测到T、B、NK细胞。此外，本研究未开展预处理方案，便对一头ART−/−IL2RG−/Y雄性SCID猪实施了完全猪白细胞抗原（SLA）匹配供体的骨髓移植，成功重建了其体内的猪T细胞与NK细胞。随后，我们向胎龄中的ART−/−IL2RG−/Y SCID猪胎儿体内注射了体外培养的分选人类CD34+脐血细胞。仔猪出生后，可在其脐血与外周血中检测到人类CD45+CD3ε+细胞；同时，在这些动物的骨髓、脾脏、肝脏、胸腺及肠系膜淋巴结中也可检测到人类白细胞。综上，本研究描述了推进免疫人源化SCID猪模型开发的关键进展。