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Genetic engineering is a promising tool to repair genetic disorders, improve graft function or to reduce immune responses towards the allografts. Ex vivo organ perfusion systems have the potential to mitigate ischemic-reperfusion injury, prolong preservation time or even rescue organ function. We aim to combine both technologies to develop a modular platform allowing the genetic modification of vascularized composite (VC) allografts. Rat hind limbs were perfused ex vivo under subnormothermic conditions with lentiviral vectors. Specific perfusion conditions such as controlled pressure, temperature and flow rates were optimized to support the genetic modification of the limbs. Genetic modification was detected in vascular, muscular and dermal limb tissues. Remarkably, skin follicular and interfollicular keratinocytes as well as endothelial cells (ECs) showed stable transgene expression. Furthermore, levels of injury markers such as lactate, myoglobin and lactate dehydrogenase (LDH) as well as histological analyses showed that ex vivo limb perfusion with lentiviral vectors did not cause tissue damage and limb cytokine secretion signatures were not significantly affected. The use of ex vivo VC perfusion in combination with lentiviral vectors allows an efficient and stable genetic modification of limbs representing a robust platform to genetically engineer limbs towards increasing graft survival after transplantation.

Patients with kidney failure depend on transplantation as the only curative option. Xenotransplantation re-emerged as a promising alternative to enlarge the available organ pool. However, the 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 the low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we immortalized PTEC (imPTEC) by overexpression of simian virus 40 T large antigen. imPTEC showed typical morphology, phenotype, and functionality, but maintained steady cell cycling rates. Furthermore, SLA class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for shRNAs targeting β2-microglobulin and the class II transactivator. This contributed to reduce xenogeneic T-cell cytotoxicity (P = 0.0069) and decrease pro-inflammatory cytokine secretion such as IL-6 and IFN-γ. This study showed the feasibility to generate highly proliferative renal tubular cells and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTEC demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.

Genetic engineering is a promising tool to repair genetic disorders, improve graft function, or reduce immune responses toward allografts.

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p

Xenotransplantation may become the highly desired solution to close the gap between the availability of donated organs and number of patients on the waiting list. In recent years, enormous progress has been made in the development of genetically engineered donor pigs. The introduced genetic modifications showed to be efficient in prolonging xenograft survival. In this review, we focus on the type of immune responses that may target xeno-organs after transplantation and promising immunogenetic modifications that show a beneficial effect in ameliorating or eliminating harmful xenogeneic immune responses. Increasing histocompatibility of xenografts by eliminating genetic discrepancies between species will pave their way into clinical application.

Introducción: El gen de fusión BCR-ABL1 está presente en al menos la cuarta parte de pacientes adultos con leucemia linfoblástica aguda de células B. Su detección determina la viabilidad del tratamiento con inhibidores de tirosina quinasas (TKIs) que se controla mediante la cuantificación de transcritos de BCR-ABL1. Algunos pacientes no responden o recaen al tratamiento debido a la presencia de mutaciones en el dominio tirosina quinasa del gen BCR-ABL1. Reporte de caso: Se reporta un paciente con BCR-ABL1 que logra una respuesta molecular luego de iniciado la terapia con imatinib; sin embargo, recae después de quince meses cambiándose el tratamiento a dasatinib. El cambio no permitió una respuesta molecular a la terapia. Retrospectivamente, se realizó la búsqueda de mutaciones en el BCR-ABL1 encontrándose tres mutaciones de resistencia a terapia (E459K, E255K y V299L). Conclusiones: La aparición de mutaciones durante el tratamiento con TKIs tiene un fuerte impacto en el progreso de la enfermedad, siendo relevante la búsqueda de mutaciones ante recaída o persistencia de transcritos del BCR-ABL1.

The fusion gene BCR-ABL1 is present in at least the fourth part of B-cell acute lymphoblastic leukemia adult cases. Patients with this fusion gene are candidates to tyrosine kinase inhibitors treatment, and the response to this therapy can be measure by quantification of BCR-ABL1 transcripts. Some patients relapse because the presence of mutations in the tyrosine kinase domain of BCR-ABL1.This is a report of a patient with BCR-ABL1 who initially achieved molecular response with imatinib therapy, relapsing after fifteen months. The treatment was changed to dasatinib, but the patient doesn't achieve molecular response. Retrospectively, we analyzed the tyrosine kinase domain of BCR-ABL1 and we found three mutations (E459K, E255K and V299L).We conclude that gain of mutations during treatment with TKIs has strong impact in the progress of disease, being relevant the detection of BCR-ABL1 mutations in relapsed patients or in case of BCR-ABL1 persistence.