CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin.

Formalin, a common laboratory fixative, is a type 1 carcinogen; a biohazard with risks, environmental, disposal, and legal costs; and a chemical modifier of protein epitopes in tissues. A less-toxic tissue preservation method is therefore badly needed. We have developed a novel tissue preservation medium, Amber, composed of low-potassium dextran glucose, 10% honey, and 1% coconut oil. This study investigates Amber as compared with formalin with respect to the following aspects: (1) histologic preservation, (2) epitope integrity with immunohistochemistry (IHC) and immunofluorescence (IF), and (3) integrity of tissue RNA. Rat and human lung, liver, kidney, and heart tissues were collected and stored for 24 hours at 4 °C in Amber or formalin. The tissues were evaluated with hematoxylin and eosin; IHC: thyroid transcription factor, muscle-specific actin, hepatocyte-specific antigen, and common acute lymphoblastic leukemia antigen; and IF: VE-cadherin, vimentin, and muscle-specific actin. RNA quality upon extraction was also assessed. Amber demonstrated superior and/or noninferior performance in rat and human tissue evaluation with respect to standard techniques of histology, IHC, IF, and extracted RNA quality. Amber maintains high-quality morphology without compromising the ability to perform IHC and nucleic acid extraction. As such, Amber could be a safer and superior substitute to formalin for clinical tissue preservation for contemporary pathological examination.

Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression.

BACKGROUND: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung. METHODS: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting. RESULTS: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients. CONCLUSIONS: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.

Pulmonary Enteric Adenocarcinoma Harboring a BRAF G469V Mutation.

Pulmonary enteric adenocarcinoma (PEAC) is a rare subtype of lung cancer that should be differentiated from colorectal cancer metastasis. Little is known about its genetic background. An 84-year-old male with adenocarcinoma of the lung underwent left upper lobectomy. The histology of the surgical specimen was suggestive of PEAC. Gastrointestinal and colorectal fiberscopy revealed no evidence of colorectal cancer. Next-generation sequencing of the tumor identified a G469V substitution in serine/threonine-protein kinase B-raf (BRAF). Based on the higher prevalence of the G469 substitution in BRAF-mutant lung adenocarcinoma than in BRAFmutant colorectal cancer, the tumor likely originated from the lung. Identification of mutational genotype may be of some help in distinguishing PEAC from the lung metastasis of colorectal cancer.

SOCS3 overexpression in T cells ameliorates chronic airway obstruction in a murine heterotopic tracheal transplantation model.

PURPOSE: Suppressor of cytokine signaling-3 (SOCS3) is a negative feedback inhibitor of cytokine signaling with T-cell-mediated immunosuppressive effects on obliterative bronchiolitis (OB). In this study, we aimed to investigate the impact of T-cell-specific overexpression of SOCS3 using a murine heterotopic tracheal transplantation (HTT) model. METHODS: Tracheal allografts from BALB/c mice were subcutaneously transplanted into wild-type C57BL/6J (B6; WT) mice and SOCS3 transgenic B6 (SOCS3TG) mice. Tracheal allografts were analyzed by immunohistochemistry and quantitative polymerase chain reaction assays at days 7 and 21. RESULTS: At day 21, allografts in SOCS3TG mice showed significant amelioration of airway obstruction and epithelial loss compared with allografts in WT mice. The intragraft expression of IFN-γ and CXCL10 was suppressed, while that of IL-4 was enhanced in SOCS3TG mice at day 7. The T-bet levels were lower in SOCS3TG allografts than in WT allografts at day 7. CONCLUSION: We revealed that the overexpression of SOCS3 in T cells effectively ameliorates OB development in a murine HTT model by inhibiting the Th1 phenotype in the early phase. Our results suggest that the regulation of the T-cell response, through the modulation of SOCS expression, has potential as a new therapeutic strategy for chronic lung allograft dysfunction.

Pneumatosis intestinalis after lung transplantation for pulmonary graft-versus-host disease.

Pneumatosis intestinalis, which could complicate a spectrum of clinical conditions ranging from benign to life-threatening, is a rarely encountered complication after lung transplantation (LT). We describe two cases in which PI developed as a complication following LT for pulmonary graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In addition to the long-term immunosuppression administered for pulmonary GVHD, the intense immunosuppression needed after LT might increase the risk of PI in lung transplant recipients after HSCT. Conservative therapy should be considered for the treatment of PI developing after LT.

[A case of bleeding stomal varices during the course of oxaliplatin-based chemotherapy for recurrent rectal cancer].

A 51-year-old man with a history of an abdominoperineal resection of the rectum and colostomy for rectal cancer underwent chemotherapy for multiple liver metastases.Twenty -two courses of the folinic acid, 5-fluorouracil(5-FU)and oxaliplatin(FOLFOX4)/bevacizumab(BEV)regimen and 39 courses of 5-FU/Leucovorin/BEV were administered.Progressive splenomegaly and stomal varices were observed during the course of chemotherapy.The patient was admitted due to excessive bleeding after colostomy.Angiography revealed bleeding stomal varices secondary to portal hypertension.Splenectomy was performed with subsequent reduction in the size of the stomal varices and no rebleeding was observed.Oxaliplatin -based chemotherapy could lead to hepatic sinusoidal dilation and induce splenomegaly and varix formation secondary to portal hypertension.Our experience with this case suggests that careful attention should be paid to stomal varices in colostomy patients receiving oxaliplatin-based chemotherapy.

[Traumatic hemothorax treated with transcatheter arterial embolization].

A 33-year-old man was transported to our hospital following a traffic accident. He was found to have hemopneumothorax, multiple rib fractures and lung injury by computed tomography(CT). Despite thoracic drainage and fluid resuscitation, he became hemodynamically unstable. At 2 hours after arrival, CT revealed worsening in hemothorax. Emergency angiography of intercostal arteries showed signs of hemorrhage from intercostal arteries, and embolization of the 3∼6th intercostal arteries was performed. After transcatheter arterial embolization(TAE), his vital signs got stable and he was discharged without significant complication.

Receptor sorting within endosomal trafficking pathway is facilitated by dynamic actin filaments.

Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs.

Fission of tubular endosomes triggers endosomal acidification and movement.

The early endosome acts as a sorting station for internalized molecules destined for recycling or degradation. While recycled molecules are sorted and delivered to tubular endosomes, residual compartments containing molecules to be degraded undergo "maturation" before final degradation in the lysosome. This maturation involves acidification, microtubule-dependent motility, and perinuclear localization. It is currently unknown how sorting and the processes of maturation cooperate with each other. Here, we show that fission of a tubular endosome triggers the maturation of the residual endosome, leading to degradation. Use of the dynamin inhibitor dynasore to block tubular endosome fission inhibited acidification, endosomal motility along microtubules, perinuclear localization, and degradation. However, tubular endosome fission was not affected by inhibiting endosomal acidification or by depolymerizing the microtubules. These results demonstrate that the fission of recycling tubules is the first important step in endosomal maturation and degradation in the lysosome. We believe this to be the first evidence of a cascade from sorting to degradation.

Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.