RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies

Remarkable progress in the experimental and clinical applications of cell-based therapies has identified stem cells and their derived products as potential candidates for regenerative therapies for many disorders. The use of autologous stem cells as source for regenerative therapeutic products is strongly limited by their low availability. Therefore, the future applications of in vitro pharmed therapeutic cell products will most likely occur in an allogeneic manner. However, the high variability of the human leukocyte antigen (HLA) represents a major obstacle to the application of off-the-shelf products. We have developed a strategy to decrease the immunogenicity of in vitro generated cell products by silencing HLA expression using RNAi. HLA expression was permanently silenced in CD34+ hematopoietic stem and progenitor cells and induced the pluripotent stem cells to generate HLA-universal cells sources, which were then used for the differentiation of low immunogenic cell products. In this chapter, we will provide an overview about an RNAi-based strategy to reduce the immunogenicity of cell-based therapies, and in particular in the generation of HLA-universal platelets and tissues

Human Amniotic Membrane: A review on tissue engineering, application, and storage

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity

Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection

Additional file 2: Figure S2. Analysis of HAdV-specific T-cell responses against the novel immunodominant T-cell epitope in healthy donors

Repeated freezing procedures preserve structural and functional properties of amniotic membrane for application in ophthalmology

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing– thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During ex vivo Organ Perfusion

Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic ex vivo perfusion system to deliver lentiviral vectors encoding shRNAs targeting β2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of β2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

Figure S1. Feasibility of cryopreservation. (A) Tricistronic IDLV encoding for hGM-CSF, hIFN-α and CMV-pp65 protein used to generate SmyleDCpp65. (B) Scheme of SmyleDCpp65 generation. Monocytes were isolated by MACS selection, pre-conditioned with cytokines for 8 h, and transduced with IDLV-G2α2pp65 for 16 h. After transduction, cells were harvested and cryopreserved at 2x106 cells/mL/vial. Cells were analyzed immediately after thaw (AT) or cultured in medium without exogenous cytokines for 7 days. (C) Viability (7AADneg) and identity (CD14 + expression level) of cell product (AT). (D) Total IDLV copy numbers detected by RT-q-PCR in the transduced cell groups AT and after 7 days in culture. (E) pp65 expression in SmyleDCpp65 (CD14neg, CD11cbright) after 7 days of in vitro culture. (F) Viability, down regulation of monocyte marker (CD14), identity (CD11cbright and HLA-DR) and functional markers (CD86 and CD80) expressed in SmyleDCpp65 7 days after in vitro culture

Oxygen carriers affect kidney immunogenicity during ex-vivo machine perfusion

Normothermic ex-vivo machine perfusion provides a powerful tool to improve donor kidney preservation and a route for the delivery of pharmacological or gene therapeutic interventions prior to transplantation. However, perfusion at normothermic temperatures requires adequate tissue oxygenation to meet the physiological metabolic demand. For this purpose, the addition of appropriate oxygen carriers (OCs) to the perfusion solution is essential to ensure a sufficient oxygen supply and reduce the risk for tissue injury due to hypoxia. It is crucial that the selected OCs preserve the integrity and low immunogenicity of the graft. In this study, the effect of two OCs on the organ's integrity and immunogenicity was evaluated. Porcine kidneys were perfused ex-vivo for four hours using perfusion solutions supplemented with red blood cells (RBCs) as conventional OC, perfluorocarbon (PFC)-based OC, or Hemarina-M101 (M101), a lugworm hemoglobin-based OC named HEMO2life®, recently approved in Europe (i.e., CE obtained in October 2022). Perfusions with all OCs led to decreased lactate levels. Additionally, none of the OCs negatively affected renal morphology as determined by histological analyses. Remarkably, all OCs improved the perfusion solution by reducing the expression of pro-inflammatory mediators (IL-6, IL-8, TNFα) and adhesion molecules (ICAM-1) on both transcript and protein level, suggesting a beneficial effect of the OCs in maintaining the low immunogenicity of the graft. Thus, PFC-based OCs and M101 may constitute a promising alternative to RBCs during normothermic ex-vivo kidney perfusion

Animal Models in Allogenic Solid Organ Transplantation

Animal models provide the link between in vitro research and the first in-man application during clinical trials. They provide substantial information in preclinical studies for the assessment of new therapeutic interventions in advance of human clinical trials. However, each model has its advantages and limitations in the ability to imitate specific pathomechanisms. Therefore, the selection of an animal model for the evaluation of a specific research question or evaluation of a novel therapeutic strategy requires a precise analysis. Transplantation research is a discipline that largely benefits from the use of animal models with mouse and pig models being the most frequently used models in organ transplantation research. A suitable animal model should reflect best the situation in humans, and the researcher should be aware of the similarities as well as the limitations of the chosen model. Small animal models with rats and mice are contributing to the majority of animal experiments with the obvious advantages of these models being easy handling, low costs, and high reproductive rates. However, unfortunately, they often do not translate to clinical use. Large animal models, especially in transplantation medicine, are an important element for establishing preclinical models that do often translate to the clinic. Nevertheless, they can be costly, present increased regulatory requirements, and often are of high ethical concern. Therefore, it is crucial to select the right animal model from which extrapolations and valid conclusions can be obtained and translated into the human situation. This review provides an overview in the models frequently used in organ transplantation research

Animal Models in Allogenic Solid Organ Transplantation

Animal models provide the link between in vitro research and the first in-man application during clinical trials. They provide substantial information in preclinical studies for the assessment of new therapeutic interventions in advance of human clinical trials. However, each model has its advantages and limitations in the ability to imitate specific pathomechanisms. Therefore, the selection of an animal model for the evaluation of a specific research question or evaluation of a novel therapeutic strategy requires a precise analysis. Transplantation research is a discipline that largely benefits from the use of animal models with mouse and pig models being the most frequently used models in organ transplantation research. A suitable animal model should reflect best the situation in humans, and the researcher should be aware of the similarities as well as the limitations of the chosen model. Small animal models with rats and mice are contributing to the majority of animal experiments with the obvious advantages of these models being easy handling, low costs, and high reproductive rates. However, unfortunately, they often do not translate to clinical use. Large animal models, especially in transplantation medicine, are an important element for establishing preclinical models that do often translate to the clinic. Nevertheless, they can be costly, present increased regulatory requirements, and often are of high ethical concern. Therefore, it is crucial to select the right animal model from which extrapolations and valid conclusions can be obtained and translated into the human situation. This review provides an overview in the models frequently used in organ transplantation research

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting β2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation