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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 within a naturally occurring genetic background. We confirmed pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one 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 injections of cultured human CD34 selected cord blood cells into the fetal SCID pigs. At birth, human CD45 CD3ε cells were detected in cord and peripheral blood of 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.

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ( ) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the gene ( ), while the other mutation is within exon 10 that results in a premature stop codon ( ). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε cells can be detected in most SCID animals. Upon further assessment, we found that , and SCID pigs had abnormally small populations of CD3ε cells, but not CD79α cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε cells within lymph nodes prior to any environmental exposure. CD3ε cells in SCID pigs appeared to have a skewed CD4α CD8α CD8β T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRβ V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients.

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a \"leaky\" Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

Swine biomedical models have been gaining in popularity over the last decade, particularly for applications in oncology research. Swine models for cancer research include pigs that have severe combined immunodeficiency for xenotransplantation studies, genetically modified swine models which are capable of developing tumors in vivo, as well as normal immunocompetent pigs. In recent years, there has been a low success rate for the approval of new oncological therapeutics in clinical trials. The two leading reasons for these failures are either due to toxicity and safety issues or lack of efficacy. As all therapeutics must be tested within animal models prior to clinical testing, there are opportunities to expand the ability to assess efficacy and toxicity profiles within the preclinical testing phases of new therapeutics. Most preclinical in vivo testing is performed in mice, canines, and non-human primates. However, swine models are an alternative large animal model for cancer research with similarity to human size, genetics, and physiology. Additionally, tumorigenesis pathways are similar between human and pigs in that similar driver mutations are required for transformation. Due to their larger size, the development of orthotopic tumors is easier than in smaller rodent models; additionally, porcine models can be harnessed for testing of new interventional devices and radiological/surgical approaches as well. Taken together, swine are a feasible option for preclinical therapeutic and device testing. The goals of this resource are to provide a broad overview on regulatory processes required for new therapeutics and devices for use in the clinic, cross-species differences in oncological therapeutic responses, as well as to provide an overview of swine oncology models that have been developed that could be used for preclinical testing to fulfill regulatory requirements.

Swine with severe combined immunodeficiency (SCID) are an emerging large animal model for biomedical research. There have been several SCID pig models described since our first discovery of naturally occurring SCID with mutations in Artemis (DCLRE1C) in 2012. SCID animals are particularly useful in biomedical research due to their lack of T, B, and sometimes NK cells. Absence of the adaptive immune system allows for human cell and tissue xenotransplantation into these SCID animals. The works described within this thesis are categorized under four main goals: (1) further characterization of the immune system of Art-/- SCID pigs, (2) development of a method for in utero injection of human stem cells, (3) in utero injection of human stem cells for the study of human immune cell engraftment within Art-/- IL2RG-/Y SCID pigs, and (4) development of an ovarian carcinoma model in SCID pigs.Characterization of the SCID pig immune system is important so that researchers can understand how components of the system could impact their research model within the pigs. This thesis describes the characterization of porcine monocytes and their interaction with human cells, as well as “leaky” T cell development in SCID pigs. Firstly, we show that porcine SIRPA, which is expressed on myeloid cells, binds to the “self” protein, CD47, on human cells. Binding between porcine SIRPA and human CD47 prevents phagocytosis of human cells by porcine myeloid cells. Binding compatibility of these two proteins suggests that porcine monocyte phagocytosis of human cells would not be a barrier to human immune cell engraftment within SCID pigs. In addition, this thesis provides an initial investigation into “leaky” CD3ε+ T cells that develop in Art-/- SCID pigs. If functional, these T cells could negatively impact the ability of human cells to engraft within the pigs. I describe that there are small populations of leaky T cells within the blood and lymph nodes of Art-/- SCID pigs.Another major goal described within this thesis is the development of in-house laparotomy surgical protocols to be utilized for the in utero injection of SCID pig fetuses with human hematopoietic stem cells. We performed two practice laparotomies on non-SCID litters and attempted to inject fetuses within the fetal liver or intraperitoneal space with saline and wire. We showed that our procedures were safe and successful, as we did not have incidence of abortion and 5 of 6 wire-injected piglets were liveborn. We were able to radiographically detect injected wire within the intraperitoneal space, and in one case, in the liver.After developing the laparotomy procedures, we were able to utilize the surgery techniques to introduce human hematopoietic stem cells into fetal SCID pigs. Art-/- IL2RG-/Y pigs were generated from an Art-/- fetal fibroblast cell line via CRISPR/Cas9 mutagenesis. These Art-/- IL2RG-/Y SCID pigs have a T- B- NK- cellular phenotype, which is optimal for humanization studies, based on previous SCID mouse literature. SCID fetuses were injected with human hematopoietic stem cells utilizing developed laparotomy procedures. We detected human leukocytes cells within the blood, thymus, spleen, liver, and bone marrow of the injected Art-/- IL2RG-/Y pigs. More specifically, we detected human CD3ε+ cells within the blood, spleen, and thymus of these animals. These findings warrant further investigation to improve the reconstitution of human cells within SCID pigs.Lastly, I describe the beginning stages of a model of ovarian cancer in SCID pigs. Human ovarian serous papillary carcinoma (OSPC)-ARK1 cells were injected into the ear and neck muscles to answer if this carcinoma could develop in an ectopic site within the SCID pig. We found that 3 out of 4 injected SCID pigs developed carcinomas in at least one injection site. OSPC-ARK1 tumors from SCID pigs, SCID mice, as well as the OSPC-ARK1 cell line and the original human tumor sample were stained with commonly used diagnostic markers: Claudin 3/4, Cytokeratin 7, p16, and EMA to assess if tumors that developed in SCID pigs resembled the human tumor. We found that expression of these proteins was highly similar between SCID pig and human carcinomas. The next step of this project is to develop an orthotopic model of ovarian cancer by injection of these cells into the ovary of SCID pigs. Development of such a model could be used for diagnostic imaging research.In all, this work describes further characterization and development of biomedical models in SCID pigs with a focus on humanization and cancer modeling. The findings described here can be utilized to improve SCID pig models in future research.

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 and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a 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 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.