• Expansion of functional personalized cells with specific transgene combinations.

      Lipps, Christoph; Klein, Franziska; Wahlicht, Tom; Seiffert, Virginia; Butueva, Milada; Zauers, Jeannette; Truschel, Theresa; Luckner, Martin; Köster, Mario; MacLeod, Roderick; et al. (Springer Nature, 2018-03-08)
      Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
    • Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

      Krooss, Simon Alexander; Dai, Zhen; Schmidt, Florian; Rovai, Alice; Fakhiri, Julia; Dhingra, Akshay; Yuan, Qinggong; Yang, Taihua; Balakrishnan, Asha; Steinbrück, Lars; et al. (Cell Press/Elsevier, 2020-01-24)
      Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.
    • Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model.

      Junge, Norman; Yuan, Qinggong; Vu, Thu Huong; Krooss, Simon; Bednarski, Christien; Balakrishnan, Asha; Cathomen, Toni; Manns, Michael P; Baumann, Ulrich; Sharma, Amar Deep; et al. (2018-02-27)
      To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-)mice by homologous-recombination-mediated targeted addition of theFahgene.
    • MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

      Yang, Dakai; Dai, Zhen; Yang, Taihua; Balakrishnan, Asha; Yuan, Qinggong; Vondran, Florian W R; Manns, Michael P; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep; et al. (Wiley, 2020-09-15)
      The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.
    • MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma

      Komoll, Ronja Melinda; Hu, Qingluan; Olarewaju, Olaniyi; von Döhlen, Lena; Yuan, Qinggong; Xie, Yu; Tsay, Hsin Chieh; Daon, Joel; Qin, Renyi; Manns, Michael P; et al. (2021-01-01)
      Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
    • Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.

      Yang, Taihua; Poenisch, Marion; Khanal, Rajendra; Hu, Qingluan; Dai, Zhen; Li, Ruomeng; Song, Guangqi; Yuan, Qinggong; Yao, Qunyan; Shen, Xizhong; et al. (Elsevier, 2021-08-25)
      Background & aims: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. Methods: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Results: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Conclusion: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Lay summary: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.