In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

Coronavirus 2019 (COVID-19) venovenous extracorporeal oxygenation: Single community hospital results and insights.

BACKGROUND: The role of extracorporeal membrane oxygenation (ECMO) for patients with refractory respiratory failure due to coronavirus 2019 (COVID-19) is still unclear even now over a year into the pandemic. ECMO is becoming more commonplace even at smaller community hospitals. While the advantages of venovenous (VV) ECMO in acute respiratory distress syndrome (ARDS) from COVID-19 have not been fully determined, we believe the benefits outweighed the risks in our patient population. Here we describe all patients who underwent VV ECMO at our center. METHODS: All patients placed on ECMO at our center since the beginning of the pandemic, May 5, 2020, until February 20, 2021 were included in our study. All patients placed on ECMO during the time period described above were followed until discharge or death. The primary endpoint was in-hospital death. Secondary outcomes included discharge disposition, that is, whether patients were sent to a long-term acute care center (LTAC), inpatient rehabilitation, or went directly home. RESULTS: A total of 41 patients were placed on VV ECMO for refractory acute respiratory failure. Survival to discharge, the primary end point, was 63.4% (26/41). Inpatient mortality was 36.6% (15/41). CONCLUSIONS: We show here that a successful high-volume VV ECMO program for ARDS is achievable at even a medium-size community hospital. We think our success can be replicated by most small- and medium-size community hospitals with cardiothoracic surgery programs and intensivist teams.

Diffuse histology-proven mucinous cystic neoplasm of the pancreas: A case report and review of literature.

INTRODUCTION: Mucinous cystic neoplasms (MCN) are rare premalignant neoplasms of the pancreas that are typically found as single lesions in the pancreatic body and tail of women in the fifth and sixth decade of life, do not communicate with the pancreatic ductal system and are characterized by mucin-producing epithelium supported by ovarian-type stroma. PRESENTATION OF CASE: We present here a case of diffuse pancreatic involvement by MCN in a 64-year-old woman with chronic pancreatitis. Pre-operative suspicion for MCN was low due to the multi-centric nature of the lesions and imaging/biochemical fluid analysis demonstrating connection with the pancreatic ductal architecture. The patient underwent total pancreatectomy with pathology showing multiple cysts lined by flat epithelium with focal ovarian-type stroma, consistent with low-grade MCN. DISCUSSION: The presence of ovarian stroma on histological analysis is one of the defining characteristics of MCNs per WHO guidelines, and is mandatory for its diagnosis. Only one case of diffuse MCN has been previously described in the literature; however, in this case the authors were not able to reach a definitive histological diagnosis based on WHO criteria. CONCLUSION: To our knowledge, this is the first report of diffuse histology-proven MCN of the pancreas.

Fetoscopy-Assisted Percutaneous Decompression of the Distal Trachea and Lungs Reverses Hydrops Fetalis and Fetal Distress in a Fetus with Laryngeal Atresia.

We present a case of prenatal hydrops secondary to congenital high airway obstruction syndrome (CHAOS) that was treated with fetoscopy-assisted needle decompression. A 22-year-old G3P2 woman presented after a 21-week ultrasound demonstrated CHAOS. The fetus developed hydrops at 25 weeks, characterized by abdominal ascites, pericardial effusion, and scalp edema. Fetal MRI showed complete obstruction of the glottis and subglottic airway, suggestive of laryngeal atresia. At 27 weeks, due to the progression of the hydrops, operative fetoscopy was proposed and performed. Fetal laryngoscopy confirmed fusion of the vocal cords and laryngeal atresia. The atretic segment was a solid cartilaginous block, preventing intubation. Using the fetoscope to stabilize the fetal head and neck, we performed ultrasound-guided percutaneous needle drainage of the cervical trachea through the anterior fetal neck. We removed 17 mL of viscous fluid from the lower trachea, resulting in immediate lung decompression. Two weeks later, ultrasound confirmed hydrops resolution. The patient was delivered and tracheostomy performed at 30 weeks via an ex utero intrapartum treatment (EXIT) procedure after progression of preterm labor. At 27 days of life, the infant was stable on minimal ventilator support. To our knowledge, this is the first successful report of an ultrasound-guided percutaneous tracheal decompression through the anterior neck of a fetus with CHAOS secondary to laryngeal atresia.

Ex Vivo Hepatocyte Reprograming Promotes Homology-Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation.

Ex vivo CRISPR/Cas9-mediated gene editing in hepatocytes using homology-directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end-joining is the predominant DNA repair pathway for double-strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9-mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down-regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo. To assess functionality long-term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single-point mutation were transduced ex vivo with two adeno-associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2-kb homology template. Adeno-associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte-directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.

Autologous Gene and Cell Therapy Provides Safe and Long-Term Curative Therapy in A Large Pig Model of Hereditary Tyrosinemia Type 1.

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson's trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector's ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models.

BACKGROUND: Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes. METHODS: Pig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography-computed tomography. Fumarylacetoacetate hydrolase-deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals' ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione. RESULTS: Positron emission tomography-computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell-transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase-positive hepatocytes in the liver, correlating with similar weight stabilization curves. CONCLUSION: Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase-deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.

Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah-/- cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah-/- mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

Concise Review: Liver Regenerative Medicine: From Hepatocyte Transplantation to Bioartificial Livers and Bioengineered Grafts.

Donor organ shortage is the main limitation to liver transplantation as a treatment for end-stage liver disease and acute liver failure. Liver regenerative medicine may in the future offer an alternative form of therapy for these diseases, be it through cell transplantation, bioartificial liver (BAL) devices, or bioengineered whole organ liver transplantation. All three strategies have shown promising results in the past decade. However, before they are incorporated into widespread clinical practice, the ideal cell type for each treatment modality must be found, and an adequate amount of metabolically active, functional cells must be able to be produced. Research is ongoing in hepatocyte expansion techniques, use of xenogeneic cells, and differentiation of stem cell-derived hepatocyte-like cells (HLCs). HLCs are a few steps away from clinical application, but may be very useful in individualized drug development and toxicity testing, as well as disease modeling. Finally, safety concerns including tumorigenicity and xenozoonosis must also be addressed before cell transplantation, BAL devices, and bioengineered livers occupy their clinical niche. This review aims to highlight the most recent advances and provide an updated view of the current state of affairs in the field of liver regenerative medicine. Stem Cells 2017;35:42-50.

Cell therapy in chronic liver disease.

PURPOSE OF REVIEW: To date, the only curative treatment for end-stage liver disease is liver transplantation, which is limited by the shortage of available organs. Cell therapy, in the form of cell transplantation or cell-based extracorporeal support devices, may in the future offer an alternative to transplantation, or at least provide liver function support as a bridging therapy until surgery may be performed. The purpose of this review is to highlight the most recent advances made in the field of cell therapy and regenerative medicine for the treatment of chronic liver disease. RECENT FINDINGS: After hepatocyte transplantation, long-term engraftment in the liver and spleen may be achieved, which can be stimulated through preconditioning, multiple infusions, and inflammatory response blockade. Mesenchymal stem cells are promising candidates for cell transplantation, as they have been shown to reduce liver fibrosis and support endogenous regeneration. Adipose tissue-derived stem cells are also being tested in this setting, because of their ready availability. Bioartificial liver devices are being built that allow for effective preservation of hepatocytes, and one such device has recently demonstrated survival benefit in a porcine model of liver failure. SUMMARY: Cell transplantation of primary hepatocytes or stem cell-derived hepatocyte-like cells for the treatment of chronic liver disease holds promise. Bioartificial liver systems may in the future be able to bridge acute-on-chronic liver failure patients to liver transplantation.