Jonathan Green - US grants

The experiments proposed in this application are designed to address the role of specific T cell accessory molecule interactions in the generation of a pulmonary inflammatory response. It has been shown in animal models of inflammatory lung disease that the T lymphocyte is critical in the recruitment of leukocytes and the initiation of the inflammatory cascade. However, interruption of effector cytokines has had only marginal success in the prevention of the development of pulmonary inflammatory diseases. Manipulation of the signals required to initiate T cell function may be a more promising approach. The initiation of T cell proliferation and effector function requires not only antigen receptor signaling, but the interaction of other receptor ligand pairs. These additional signals have been termed costimulatory signals. CD28 has been demonstrated to be a bona fide costimulatory interaction in both in vitro and in vivo studies. Preliminary studies investigating the T cell function of CD28-deficient mice has led to the identification of CD43 as a potential important mediator of both T cell activation and cell death. The specific aims are l) Determine the basis for the differential induction of proliferation or cell death following CD43 ligation. 2) Determine if the absence of CD43 signaling alters the outcome of an immune response in vitro, and 3) Examine the role of T cell costimulatory molecules CD43 and CD28 in the generation of a pulmonary inflammatory response. Specific inhibitors of these interactions and transgenic animals will allow for the careful evaluation of the contribution of these costimulatory interactions in the development of inflammatory lung disease. These studies have the potential for leading to new,specific treatments for immune mediated pulmonary disease. The work described above will be complemented by didactic training in several formats. Washington University has an extensive seminar series in both immunology and pulmonary medicine in which Dr. Green will be an active participant. Enrollment in formal course work on advanced topics of immunology and molecular biology will further enhance the training received under this award. The availability of internationally recognized researchers in both immunology and pulmonary medicine provide an ideal environment for the accomplishment of the research goals proposed in this application.

The primary goal of this proposal is to determine the mechanism by which CD43 regulates an inflammatory response in the lung. CD43 is a glycoprotein that is highly expressed on a variety of inflammatory cells including T cells, monocytes and neutrophils. We have demonstrated that ligation of CD43 augments the response of T cells to antigenic challenge. In addition, our study of CD43 deficient mice has shown that T cells from these mice are hyperresponsive both in vivo and in vitro. Furthermore, lung neutrophil recruitment is augmented in CD43 deficient mice following a single intra-tracheal challenge with LPS. These data point to a critical role for CD43 in the overall regulation of inflammatory processes. We hypothesize that unique structural features of CD43 allow it to function as a barrier to interactions between cell surface receptors and their ligands, either soluble or cell bound. We propose that a novel regulatory mechanism, the proteolytic cleavage of CD43, is a critical element in the function of this protein. To test this hypothesis and determine the precise mechanism of CD43 function, we propose the following specific aims: 1) Determine the role of proteolytic cleavage of CD43 in T cell activation and proliferation and 2) Determine the structural basis of CD43 function. In specific aim 1 we propose a definitive analysis of CD43 proteolysis in the activation of primary T cells. We have developed new reagents to allow for the detection of the detection of the multiple isoforms of CD43. Utilizing both normal and TCR transgenic mice we will determine whether CD43 ligation directly inducers its loss from the cell surface, and under what circumstances in normal T cell responses this occurs. In specific aim 2 we propose to undertake a detailed molecular analysis of the structural requirements that are essential for CD43 function. By the construction of mutant forms of CD43 and expression of them on a CD43 deficient background we will gain crucial information as to the mechanism of CD43's ability to modulate T cell activation specifically, and inflammation generally. The information obtained from these studies will further our overall understanding of the factors that control inflammatory responses that allow for the rational design of novel therapeutic strategies to treat a variety of inflammatory conditions.

DESCRIPTION: (Adapted from the Applicant's Abstract): T lymphocytes are a major component of the inflammatory response and aberrant regulation of T cell activation and differentiation may be central to the pathogenesis of asthma. The CD28 co-stimulatory receptor is a critical regulator of T cell activation and differentiation, and thus may be an important target for therapy of immune mediated diseases. Manipulation of this receptor has been shown to alter the course of several animal models of disease, inducing models of antigen-induced airway inflammation. Accordingly, the primary goal of this proposal is to increase our understanding of the cellular and molecular basis by which CD28 modulates the T cell response to antigen. CD28 regulates multiple aspects of T cell functions, including proliferation, adhesion, T helper cell phenotype development and cell survival. The PI demonstrates that CD28 is essential for the development of antigen-induced inflammation in a murine model of airway disease. Sensitized CD28-deficient mice fail to develop airway inflammation or eosinophilia in response to antigen challenge. Examination of T helper cell phenotype in CD28 -/- mice demonstrates a defect in Th2 cytokine gene expression. The mechanism by which CD28 regulates these diverse aspects of T cell function is poorly understood, but likely involves multiple signaling pathways. Studies in transformed cell lines have implicated specific domains in the cytoplasmic trail of CD28 as important in signaling, but no consensus exists as to what is required for CD28 function in primary cells or in vivo responses. The data in primary T cells demonstrates a requirement for specific proline mediated interactions with the non-receptor tyrosine kinase lck in the regulation of T cell proliferation by CD28. The PI hypothesizes that multiple distinct structural domains of CD28 modulate specific features of T cell activation and differentiation. To address this, the following specific aims have been proposed: 1) determine the structural features of CD28 required for co-stimulation of primary T cells in vitro; and 2) characterize the specific cellular and molecular determinants by which CD28 regulates airway inflammation in vivo. These studies will provide critical data as to the regulation of T cell directed immune responses, and provide a rational basis for the development of new therapeutic strategies in the treatment of inflammatory lung disease.

[unreadable] DESCRIPTION (provided by applicant): Host defense against pulmonary pathogens requires the generation of effective T cell directed immune responses. T cell activation is a multi-step process that involves the coordinate participation of adhesion receptors. These interactions regulate lymphocyte trafficking to secondary lymphoid organs and to sites of inflammation, as well as the cognate interactions required for T cell recognition of antigen. The sialoglycoprotein CD43 is among the most highly expressed proteins on the surface of the T cell. CD43 has been implicated in regulating several aspects of T cell biology, including proliferation, adhesion and survival. The most striking aspect of CD43 is its ability to alter cell adhesion, which is likely the primary function of CD43. The mechanism by which CD43 regulates the adhesive properties of the T cell has been proposed to be a function exclusively of the structure of the extracellular domain. The ectodomain of CD43 is heavily glycosylated and sialylated, and as such has a rigid conformation that extends outward from the cell membrane a distance far greater than the T cell receptor. This has led to the predominant model of CD43 function, that it sterically hinders the ability of integrins and other cell surface receptors to bind ligand.Our studies demonstrate 2 critical findings that fundamentally alter the way in which CD43 must be considered. First, we have determined the cytoplasmic domain is both necessary and sufficient for the regulation of cell adhesion. Thus, the steric hindrance model is no longer a tenable explanation for how CD43 regulates cell adhesion. Second, we demonstrate that the cytoplasmic tail of CD43 contains a nuclear localization signal that can mediate translocation of the intracellular domain into the nucleus, and that CD43 can associate with known nuclear proteins. Thus, we hypothesize that the mechanism by which CD43 regulates cell adhesion is based upon specific interactions with intracellular signaling pathways, and involves a novel mechanism in which the intracellular domain translocates to the nucleus and regulates gene transcription. In this application we propose 3 specific aims to determine the mechanism by which CD43 regulates T cell function. First, we will determine the specific element(s) within the intracellular domain of CD43 that regulate cell adhesion. Both in vitro and in vivo systems will be utilized to study the effects 01 specific mutations on the adhesive properties of the cell. Second, using both a genetic and proteomic approach, we will determine the proteins that interact with the intracellular domain of CD43. This data will identify the pathways by which CD43 exerts its regulatory function. Third, we will determine how CD43 interfaces with known cytoskeletal and signaling pathways that regulate cell adhesion, and explore the novel observation that the intracellular domain of CD43 may enter the nucleus and directly alter gene transcription. The successful completion of these studies will increase our understanding of how T cells function, and suggest novel strategies to alter the outcome of T cell mediated diseases.

DESCRIPTION (Provided by the applicant): Loss of CD4+ T cell number or function may render individuals susceptible to opportunistic infection such as P. carinii pneumonia (PCP). This is exemplified by Human Immunodeficiency Virus infection, but also complicates other diseases in which normal immune cell function is perturbed. Although both observational and experimental data indicate that the CD4+ T cell is a critical determinant of the outcome of infection with Pneumocystis, the precise manner in which this effect is exerted is less clear. In addition to direct effector function, CD4+ T cells influence the function of other cells such as B cells, macrophages and cytolytic T cells through both soluble mediators as well as via direct intercellular interactions. Derangement in any of these elements of the immune response may lead to increased susceptibility to opportunistic infection. T cell activation is determined not only by engagement of the T cell antigen receptor, but also by theengagement of other receptors, termed costimulatory receptors. CD2 and CD28 are two well-described costimulatory molecules. Mice deficient in CD2 have little discernible phenotype on examination of T cell function. T cells from CD28-deficient mice have reduced proliferative responses and cytokine secretion. as well as impaired survival, but previously have not been noted to be susceptible to opportunistic infection. We have generated mice deficient in both CD2 and CD28. These mice have a profound defect in T cell activation despite normal lymphocyte numbers and distribution. Furthermore, the CD2/CD28 double deficient mice spontaneously develop and succumb to infection with P. carinii. Mice deficient only in CD28 do not develop P. carinii following co-housing with infected animals, yet are susceptible if inoculated intratracheally. The defined genetic defect that results in defective T cell function despite normal T cell number provides us with a novel opportunity to examine the role of T cells response to P. carinii. Furthermore, the differential susceptibility of CD28-deficient mice to naturally acquired infection vs direct inoculation allows us to examine how route of infection influences the host response. To address this, we propose the following two specific aims: 1 ) Characterize the response of costimulation deficient mice to infection with Pneumocystis carinii. The experiments proposed in this aim will provide quantitative data defining the susceptibility of the costimulation deficient mice and the characteristics of the immune response mounted against them. 2) Determine if reconstitution of specific cellular elements enable clearance of P. carinii infection in costimulation deficient mice. In this aim we will restore specific aspects of the host response by adoptive transfer into the costimulation deficient mice to dissect what required elements of the host response are lacking in the costimulation deficient mice. Although the observation of severe P. carirzii pneumonia in the double knockout mice was unexpected, these mice provide us with a powerful tool to examine the role of costimulatory molecules in defense against this important opportunistic pathogen.

DESCRIPTION (provided by applicant): T cell responses are highly regulated to assure appropriate and effective responses to infectious and inflammatory stimuli. A key component of this is the B7:CD28 receptor family, consisting of activating (CD28 and ICOS) and inhibitory (CTLA-4, PD-1 and BTLA) members and their ligands (B7 proteins). These receptors play a critical role in regulating lung inflammation by determining whether an immune response is initiated, and once initiated, its resolution. The goal of the studies proposed in this application is to define novel mechanisms by which the B7:CD28 family regulates the initiation and resolution of lung inflammation, and determine how this can be manipulated to therapeutic advantage. To study the molecular mechanisms controlling the initiation of inflammation, we developed gene targeted CD28-mutant knockin mice. Using these we established which signaling motifs mediate specific in vitro and in vivo CD28-dependent functions. In contrast to the prevailing dogma that PI3 kinase signaling initiated by the proximal tyrosine (Y170) motif is the critical pathway activated by CD28, we demonstrated that it is in fact dispensable for CD28 function. However, we showed that the distal proline (PYAP) motif initiated a non- redundant signaling pathway required for normal CD28-dependent responses. Despite mutation of this motif, some function remains the biochemical basis for which is unknown. Therefore, we hypothesize that novel pathways activated independent of the distal proline motif regulate CD28-dependent T cell proliferation and cytokine secretion. We further found that signaling initiated by the distal proline motif is essential for T cell: B cell collaboration and T-dependent antibody production. The signaling pathways that mediate these responses will be determined in specific aim 1 of this proposal. Ongoing inflammation can be inhibited by therapeutic manipulation of the B7:CD28 pathway using the recently approved drug CTLA4Ig, (abatacept, OrenciaTM). This drug binds to B7-1 (CD80) and B7-2 (CD86), and has been presumed to function by preventing CD28 engagement on the T cell, thereby preventing a CD28-mediated signal to the T cell. While this mechanism is true for initial T cell activation, we have established that in fact CTLA4Ig inhibits effector responses by a novel CD28-independent, inducible nitric oxide synthase (NOS2)-dependent mechanism. We further show that IFN3/STAT1 signaling is central in this response. We hypothesize that B7 engagement by CTLA4Ig induces INF3 dependent activation of macrophages or myeloid derived suppressor cells (MDSCs), and that these inhibit allergic lung inflammation by a NOS2-dependent mechanism. In this application, we propose cellular, molecular and in vivo studies to determine the mechanism of the novel mode of action for CTLA4Ig., which may have broad applicability to therapeutic manipulation of inflammatory lung disease

The host response to antigenic challenge requires initiation, effector, and resolution phases of inflammation.[unreadable] Just as positive signals are required to initiate immune responses, the resolution of inflammation is also an[unreadable] active process that depends upon signaling through inhibitory receptors. These signals are provided to T[unreadable] cells by activating or inhibitory members of the B7:CD28 receptor family. Atopic asthma is a T cell mediated[unreadable] immune response characterized by T helper 2 type inflammation in the airway. Using a murine model of this[unreadable] disease we have shown that costimulation through CD28 is important in the initiation and maintenance of[unreadable] inflammation. We now find that inhibitory receptor function is equally critical in the regulation of airway[unreadable] inflammation. We demonstrate that there is temporally regulated induction of both PD-1 and BTLA and their[unreadable] ligands in lung cells following allergen challenge. Furthermore, mice deficient in these receptors demonstrate[unreadable] prolonged airway inflammation following a single allergen challenge. Therefore, we hypothesize that the[unreadable] regulated expression of BTLA and PD-1 on lymphocytes and their ligands HVEM, PDL1 and/or PDL2[unreadable] parenchyma! or infiltrating immune cells limit the development of chronic airway inflammation. In[unreadable] Specific Aim 1 we will focus on the regulation of ligand expression. We will determine specifically which cell[unreadable] types in the lung express each ligand and what factors regulate their expression. Using an inducible[unreadable] transgenic mouse system we will determine if overexpression of the ligand in vivo can either prevent the[unreadable] onset of lung inflammation, or terminate an ongoing inflammatory response. In addition, we will use primary[unreadable] cultured murine tracheal epithelial cells to dissect the elements that regulate expression in airway cells. In[unreadable] Specific Aim 2 we turn our focus to the T cell. Our preliminary data establishes that in the absence of PD-1[unreadable] or BTLA there is prolonged inflammation following a single allergen challenge. We will extend this[unreadable] observation to determine the role of these receptors in models of chronic allergen exposure. Furthermore,[unreadable] we will determine the contributions of cell recruitment, proliferation and or survival in the accumulation of[unreadable] lymphocytes in the lung. While aims 1 and 2 we use an experimental murine model, in aim 3 we will begin[unreadable] an analysis of these receptorligand interactions in humans. We will compare the expression and function of[unreadable] both PD-1 and BTLA in normal and asthmatic subjects. Within the context of this collaborative program we[unreadable] will interact extensively with Project 1 in our studies of epithelial cells. In project 2, the focus is on regulatory[unreadable] T cells, which may utilize inhibitory receptors such as PD-1 and BTLA to regulate T cell function. Thus, the[unreadable] synergistic interactions of the group will undoubtedly strengthen the overall group. We believe the[unreadable] combination of expertise and reagents available in this program leave us uniquely poised to delineate how[unreadable] PD-1 and BTLA limit lung inflammation. Studies such as these may provide the basis for the development of[unreadable] novel immunotherapeutic approaches to chronic inflammatory lung diseases such as asthma.

DESCRIPTION (provided by applicant): Over 200,000 people die annually in the US from the consequences of sepsis. While molecularly guided therapy has been introduced for many diseases, such interventions remain elusive in the treatment of sepsis. Many patients that survive the acute phase of sepsis develop secondary infections and succumb to these later complications. The majority of septic patients are mechanically ventilated, and sepsis is an independent risk factor for ventilator associate pneumonia (VAP). VAP is the most common nosocomial infection in the ICU, is responsible for the majority of ICU antibiotic use and carries a mortality of close to 40%. While factors that interrupt normal upper airway defense mechanisms are important contributors, host immune factors likely play an equally critical role. The identification of these factors is a major goal this proposal. Importantly, these pathways may offer novel opportunities for therapeutic manipulation. Recent work has documented a state of relative immunosuppression occurring in both animal models and humans with sepsis. Apoptotic cell death of lymphocytes as well as a defect in the function of the remaining cells has been demonstrated, of which the underlying mechanism has not been determined. Prevention or reversal of this immunosuppression would be predicted to prevent many of the later sequelae of sepsis and thereby improve patient survival. During a normal immune response lymphocyte function is restricted by specific receptor mediated, inhibitory signaling pathways and by the action of distinct cell types including regulatory T (Treg) cells and myeloid derived suppressor cells (MDSC). We hypothesize that in sepsis there is an inappropriate, widespread induction of suppressive pathways resulting in the active inhibition of T cell function. We propose that increased expression of inhibitory receptors on peripheral blood lymphocytes and/or an increase in suppressor cell populations (Tregs and MDSCs) will identify and differentiate those patients at increased risk of secondary infection from those not at risk, and thereby serve as a "biomarker" allowing for targeted therapies. We also propose that the susceptibility to VAP is driven by the parenchymal cells of the lung. By expressing specific inhibitory ligands and metabolic pathways such as indoleamine 2,3 dioxygenase (IDO), the lung parenchyma creates an immunosuppressive environment that actively inhibits lymphocyte function. We propose to test this hypothesis by characterizing the blood and lung cells from with patients sepsis that develop VAP admitted to the ICU's at Barnes-Jewish Hospital. While exploratory, these studies have the potential to be paradigm shifting and lay the groundwork for novel therapeutic interventions in sepsis. Currently biologics that manipulate CTLA4, PD-1 and IDO signaling are in clinical trials as immune modulating agents in cancer patients. The results of this study will allow for the clinical identification of immunosuppressed patients at risk for infection, as well as provide preliminary data in support of a larger, definitive trial with the eventual goal of a randomized intervention trial. PUBLIC HEALTH RELEVANCE: This proposal aims to understand the reason for the suppressed immune response in critically ill patients. The relative immunosuppressed state in these patients often results in infections that are the cause of death. The studies we propose in this application will allow us to identify patients at risk for infection, and design treatments to enhance their immune function.

DESCRIPTION (provided by applicant): Sepsis is a systemic response to severe infection with an associated mortality of approximately 30%. Although many sepsis-induced deaths take place during the initial hyperinflammatory phase, those patients that survive often develop and may succumb to secondary infections. These infections are frequently caused by organisms not typically pathogenic in immunocompetent hosts, suggesting that this later phase of sepsis is characterized by clinically important immunosuppression. Although a number of mechanisms of immunosuppression have been identified in animal models of sepsis, few systematic studies have been conducted in patients. In addition, relatively new and potent mechanisms involving expression of inhibitory receptors such as PD-1, BTLA and CTLA4, as well as regulatory cell populations have been identified. Of particular interest, patients with chronic viral infection hav been demonstrated to develop a phenomenon termed immune cell exhaustion. Characterized by persistent, elevated expression of inhibitory receptors, exhausted T cells manifest severe functional defects. Supported by our preliminary data, we hypothesize that the immunosuppression in sepsis results from the combined consequences of an expansion of suppressive cells and a state of immune cell exhaustion that develops progressively over time, such that at early stages it is both identifiable and reversible. The goals of this project are to ) determine the mechanism of immunosuppression in patients that die of sepsis, and 2) identify clinically relevant markers that identify those patients with sepsis that are immunosuppressed and for which immunomodulating therapies may be of benefit. To achieve these goals, we will perform a detailed functional, phenotypic and mechanistic analysis of immune cells isolated following a novel, rapid bedside autopsy of patients that die of sepsis. This will be coupled with a prospective study of patients hospitalized in the intensive care units with sepsis, to determine clinically useful markers that can identify patients that are more severely immune compromised and therefore at increased risk for secondary infection. Together, these studies will substantially advance our understanding of the pathogenesis of this disease and directly impact the care of patients with sepsis.

DESCRIPTION (provided by applicant): Somatic cell nuclear transfer (SCNT) is an inefficient process. Our goal is to improve the development of SCNT embryos by driving fibroblast metabolism toward blastomere metabolism before and after SCNT as a way to improve development of the cloned embryo. Our overarching thesis is: Fibroblasts prior to SCNT or embryos after SCNT, driven pharmacologically toward a blastomere metabolism will make the transition from fibroblast to blastomere metabolism less dramatic and result in improved development of the embryo. Our ideal methodology would be to treat the cells to induce a blastomere-like metabolism beginning after genetic modification, or alternatively the day before SCNT. If it is necessary to begin the treatment during selection for the genetic modification, then we must confirm their ability to be expanded, form colonies and survive cryopreservation. The Specific Aims will be to: 1) Characterize treatment of the donor cells to be more blastomere- like, 2) Treat the donor cells prior to SCNT, 3) Treat the SCNT embryos only, 4) Treat the donor cells and/or the SCNT embryos, and evaluate development to term. Hypothesis 1: Fibroblast cells can be driven toward a blastomere-like (WE) metabolism. Fibroblast cells will be treated with several compounds/conditions, individually and in combination, that would drive them toward a blastomere or Warburg pathway. Hypothesis 2: Fibroblasts with a blastomere metabolism will result in SCNT embryos that have more of an embryo metabolism than those derived from control fibroblasts, and will have better post-attachment development. The treatment from Hypothesis 1 that results in the most blastomere-like metabolism will be used to treat donor cells to create SCNT embryos. Hypothesis 3: SCNT embryos induced to have a blastomere-like metabolism will develop better than control embryos. These experiments will determine if treatment of the SCNT embryo will induce a more blastomere-like metabolism and develop at a higher rate to post-attachment stages. Hypothesis 4: Fibroblasts and SCNT embryos driven to a blastomere-like metabolism will result in SCNT embryos that have more developmental competence than controls. This experiment will combine the efforts of treating the donor cells to become more blastomere-like with the treatment that enhances development of SCNT-treated embryos. In this experiment donor cells will be treated or used as controls (based on Hypothesis 2 results). In addition, SCNT embryos will be treated or used as controls (based on Hypothesis 3 results). Thus we propose a 2X2 factorial treatment structure. Embryos will be cultured to the blastocyst stage and evaluated or transferred. Endpoints will be identical to Hypothesis 3. Overall we expect that adjusting the metabolism of the donor cell prior to SCNT and/or the embryo after SCNT will result in improved development between the blastocyst stage and day 35 of development.

? DESCRIPTION (provided by applicant): Median graft survival for recipients of lung transplants remains approximately 5 years, the worst of all solid organ transplants. The major cause of death after the first year post-transplantation is chronic rejection, manifest primarily as bronchiolitis obliterans syndrome (BOS). Therefore, there is a critical unmet need for new treatment strategies that prevent or limit the progression of BOS and will thereby effectively prolong graft survival. Extracorporeal photopheresis has shown promise in the treatment of BOS, however it is typically used as a salvage therapy, instituted only after conventional approaches have failed. Initially developed for the treatment of cutaneous T cell lymphoma, ECP has also been successfully used to treat a number of diseases, including cardiac allograft rejection and chronic graft versus host disease following allogeneic stem cell transplantation. However, our ability optimally utilize this treatment is severely hampered by a lack of mechanistic understanding. The major goals of this proposal are to develop a murine model of ECP in lung transplantation, and to determine the mechanism by which ECP modulates the immune response. To accomplish this, we have developed in vitro and in vivo model systems which recapitulate the key effects of ECP, as well as the essential features of bronchiolitis obliterans. In specific aim 1, we will use an established murine orthotopic lung transplant model, pioneered by Washington University researchers, to determine the effectiveness of ECP to prevent the development of obliterative airways disease. This system will be the first, animal model to investigate ECP in lung transplantation. In specific aim 2, we will use both in vitro and in vivo approaches to dissect the mechanism by which ECP modulates effector T cell function. If successful, we will have established the first in vivo murine model system of the treatment of chronic lung allograft rejection with ECP, allowing us to explore mechanism, and most importantly, to determine how best to use ECP in the prevention and treatment of BOS in lung transplant recipients. The data obtained from this pilot project will provide essential information that will guide future studies in both mice and humans, and may provide critical supportive evidence for the early use of ECP to prevent bronchiolitis obliterans.

Project Summary -Genome Editing Testing Section The role of the Genome Editing Testing Section will be to validate unique reporter models created by the Animal Model Production Section and to test novel delivery methods and reagents provided by other members of the Somatic Cell Genome Editing (SCGE) Consortium. Additionally, the Genome Editing Testing Section will be responsible for creating detailed standard operating procedures (SOPs) and comprehensive reports for testing new genome editing technologies. It is essential to have a well- established pipeline for the validation and testing of new genome editing technology to facilitate proper refinement of the tools while ensuring the safety of the technology. The Genome Editing Testing Section will streamline the testing process while maintaining a comprehensive and rigorous comparative analysis of new genome editing tools and delivery systems. Our group is ideally suited to provide this testing due to our unique composition of individuals that are experienced with pig production, swine biomedical models, operate successful labs that routinely test cutting-edge genome editing technology, and are associated with world-renowned National Swine Resource and Research Center (NSRRC). The Genome Editing Testing Section will have a significant impact on the overall MU Swine Testing Center for Genome Editing Technologies? ability to accurately assess new technologies and further facilitate the ability to use genome editing in therapeutic circumstances.