FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3024382227> ?p ?o ?g. }

• W3024382227 endingPage "1201" @default.
• W3024382227 startingPage "1190" @default.
• W3024382227 abstract "Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3′ untranslated region (3′ UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability. Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3′ untranslated region (3′ UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability. Nowadays, gene therapy is gradually emerging as one of the most powerful strategies in the treatment of numerous inherited and acquired disorders, including hemophilia, spinal muscular atrophy, or Leber’s hereditary optic neuropathy (reviewed in Wang et al.1Wang D. Tai P.W.L. Gao G. Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378Crossref PubMed Scopus (764) Google Scholar). Even though various transgene delivery methods were developed, it appears that vectors based on adeno-associated viruses (AAVs) are the most successful so far, providing high expression of the therapeutic gene with very limited immune response in the host organism.2Naso M.F. Tomkowicz B. Perry 3rd, W.L. Strohl W.R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.BioDrugs. 2017; 31: 317-334Crossref PubMed Scopus (547) Google Scholar Still, the development of a therapy restricted to the selected organ or cell type remains challenging in many cases. Although partial selectivity of the expression can be obtained through the utilization of appropriate AAV serotype with tropism toward particular tissues,3Zincarelli C. Soltys S. Rengo G. Rabinowitz J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.Mol. Ther. 2008; 16: 1073-1080Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar it is not sufficient to prevent all off-target effects associated with the systemic delivery of the vector. Novel approaches aiming to overcome this issue focus on the engineering of cell-type-specific AAV capsids using directed evolution strategy; however, generation of such novel vectors with sufficiently high infectivity is very laborious and problematic in terms of specificity toward given cells and species.4Büning H. Srivastava A. Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors.Mol. Ther. Methods Clin. Dev. 2019; 12: 248-265Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Sometimes it is possible to administer the vector directly to the chosen tissue, i.e., into the cardiac muscle using a catheter-based electro-mechanical mapping and injection (NOGA) system.5Hartikainen J. Hassinen I. Hedman A. Kivelä A. Saraste A. Knuuti J. Husso M. Mussalo H. Hedman M. Rissanen T.T. et al.Adenoviral intramyocardial VEGF-DΔNΔC gene transfer increases myocardial perfusion reserve in refractory angina patients: a phase I/IIa study with 1-year follow-up.Eur. Heart J. 2017; 38: 2547-2555Crossref PubMed Scopus (90) Google Scholar Nevertheless, this route of delivery is invasive and can be hazardous to the patient, especially considering coexisting disorders. Thus, the most desirable cardiac gene therapy vector would be infused intravenously and show efficient and selective gene transfer to the heart. The majority of cardiac-muscle-targeting vectors are based on AAV serotype 9 (AAV9) because it is the most efficient vector in in vivo applications with described tropism toward murine heart, skeletal muscles, and liver following systemic administration.6Inagaki K. Fuess S. Storm T.A. Gibson G.A. Mctiernan C.F. Kay M.A. Nakai H. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8.Mol. Ther. 2006; 14: 45-53Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar Thus, in order to further improve the vector properties and increase its cardiac specificity, additional regulatory mechanisms should be introduced. Unfortunately, incorporation of heart-specific promoters such as alpha myosin heavy chain (α-MHC),7Gulick J. Subramaniam A. Neumann J. Robbins J. Isolation and characterization of the mouse cardiac myosin heavy chain genes.J. Biol. Chem. 1991; 266: 9180-9185Abstract Full Text PDF PubMed Google Scholar myosin light chains (MLC2v),8Su H. Joho S. Huang Y. Barcena A. Arakawa-Hoyt J. Grossman W. Kan Y.W. Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts.Proc. Natl. Acad. Sci. USA. 2004; 101: 16280-16285Crossref PubMed Scopus (96) Google Scholar and cardiac troponin T (cTnT)9Wang G. Yeh H.I. Lin J.J. Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene.J. Biol. Chem. 1994; 269: 30595-30603Abstract Full Text PDF PubMed Google Scholar into the expression cassette does not prevent vector leakage and transgene expression in other transduced tissues, e.g., skeletal muscles or liver.10Lee C.J. Fan X. Guo X. Medin J.A. Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease.J. Cardiol. 2011; 57: 115-122Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,11Pacak C.A. Sakai Y. Thattaliyath B.D. Mah C.S. Byrne B.J. Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice.Genet. Vaccines Ther. 2008; 6: 13Crossref PubMed Scopus (71) Google Scholar Other shortcomings of such a strategy may include a decreased level of transgene expression as compared with the strong promoters of viral origin like cytomegalovirus (CMV).11Pacak C.A. Sakai Y. Thattaliyath B.D. Mah C.S. Byrne B.J. Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice.Genet. Vaccines Ther. 2008; 6: 13Crossref PubMed Scopus (71) Google Scholar Hybrid promoters consisting of parts of CMV and eukaryotic promoters may help to overcome this issue; but on the other hand, such an approach may result in compromised specificity toward cardiomyocytes when compared with cardiac-specific promoter alone.12Müller O.J. Leuchs B. Pleger S.T. Grimm D. Franz W.-M. Katus H.A. Kleinschmidt J.A. Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors.Cardiovasc. Res. 2006; 70: 70-78Crossref PubMed Scopus (131) Google Scholar Generation of synthetic promoters combined with cardiomyocyte-specific transcriptional cis-regulatory motifs allows for significant improvement of transgene expression in the heart, but a strong off-target effect was visible in the skeletal muscles.13Rincon M.Y. Sarcar S. Danso-Abeam D. Keyaerts M. Matrai J. Samara-Kuko E. Acosta-Sanchez A. Athanasopoulos T. Dickson G. Lahoutte T. et al.Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy.Mol. Ther. 2015; 23: 43-52Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar As an alternative to the above-mentioned approaches of targeting transgene expression with cell-specific promoters, negative regulation exploiting tissue specifically expressed miRNAs can be utilized. Incorporation of target sites (TSs) for chosen miRNA into the 3′ untranslated region (3′ UTR) of the expression cassette enables posttranscriptional regulation of transgene expression in a cell-specific manner.14Selbach M. Schwanhäusser B. Thierfelder N. Fang Z. Khanin R. Rajewsky N. Widespread changes in protein synthesis induced by microRNAs.Nature. 2008; 455: 58-63Crossref PubMed Scopus (2784) Google Scholar Thanks to RNA interference machinery, if a given miRNA is present in the cells, vectors carrying corresponding TSs are silenced due to degradation of the transgene mRNA or inhibition of translation process.15Brown B.D. Gentner B. Cantore A. Colleoni S. Amendola M. Zingale A. Baccarini A. Lazzari G. Galli C. Naldini L. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state.Nat. Biotechnol. 2007; 25: 1457-1467Crossref PubMed Scopus (460) Google Scholar Such strategy was successfully applied for redirecting the tropism of oncolytic viruses16Dhungel B. Ramlogan-Steel C.A. Steel J.C. Synergistic and independent action of endogenous microRNAs 122a and 199a for post-transcriptional liver detargeting of gene vectors.Sci. Rep. 2018; 8: 15539Crossref PubMed Scopus (8) Google Scholar and numerous other studies, including cell lineage tracking,17Brown B.D. Venneri M.A. Zingale A. Sergi Sergi L. Naldini L. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer.Nat. Med. 2006; 12: 585-591Crossref PubMed Scopus (394) Google Scholar detargeting of transgene expression from antigen-presenting cells,18Brown B.D. Cantore A. Annoni A. Sergi L.S. Lombardo A. Della Valle P. D’Angelo A. Naldini L. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice.Blood. 2007; 110: 4144-4152Crossref PubMed Scopus (211) Google Scholar or restricting the expression to specific cell types, e.g., interneurons.19Keaveney M.K. Tseng H.A. Ta T.L. Gritton H.J. Man H.-Y. Han X. A MicroRNA-Based Gene-Targeting Tool for Virally Labeling Interneurons in the Rodent Cortex.Cell Rep. 2018; 24: 294-303Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar In case of cardiac muscle, utilization of three tandem repeats of TSs for miRNA-122 (highly expressed in the liver) and genetically engineered TSs for miRNA-206 (referred to as muscle-specific miRNA) in AAV9 vector resulted in efficient transgene delivery to the heart with concomitant repression of its expression in liver and skeletal muscles.20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In that study, male mice of the BALB/c strain were used, and neither miRNA-122 nor miRNA-206 was expressed in the hearts of these animals.20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Here, we aimed to utilize similar, miRNA-122 TS- and mutated miRNA-206 TS-containing self-complementary AAV9 vector (scAAV9-HO1-TS) for cardiac-specific expression of heme oxygenase-1 (HO-1; HMOX1) gene. We and others have shown that this stress-inducible enzyme involved in heme catabolism exerts important cardioprotective effects after ischemic injury.21Hinkel R. Lange P. Petersen B. Gottlieb E. Ng J.K.M. Finger S. Horstkotte J. Lee S. Thormann M. Knorr M. et al.Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.J. Am. Coll. Cardiol. 2015; 66: 154-165Crossref PubMed Scopus (55) Google Scholar, 22Otterbein L.E. Foresti R. Motterlini R. Heme Oxygenase-1 and Carbon Monoxide in the Heart: The Balancing Act Between Danger Signaling and Pro-Survival.Circ. Res. 2016; 118: 1940-1959Crossref PubMed Scopus (135) Google Scholar, 23Tomczyk M. Kraszewska I. Szade K. Bukowska-Strakova K. Meloni M. Jozkowicz A. Dulak J. Jazwa A. Splenic Ly6Chi monocytes contribute to adverse late post-ischemic left ventricular remodeling in heme oxygenase-1 deficient mice.Basic Res. Cardiol. 2017; 112: 39Crossref PubMed Scopus (28) Google Scholar First, the functionality of scAAV9-HO1-TS vector was confirmed in vitro. However, after its systemic administration to the animals lacking HO-1 (HO-1 knockout [KO]; C57BL/6J × FVB strain), there was almost complete repression of transgene expression not only in the liver and skeletal muscles but also in the heart. We found that this was related to the presence of miRNA-122 in the hearts of these animals. What is more, we detected this miRNA not only in the hearts of other commonly used mouse strains but also of patients suffering from various cardiomyopathies and in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). Importantly, transduction of human iPSC-derived cardiomyocytes with miRNA-122-regulated AAV, similarly to animal studies, resulted in considerable reduction of transgene expression. In order to construct scAAV9 vectors for heart-specific overexpression of a chosen transgene, we have cloned human HO-1 coding sequence under the control of the CMV promoter into the previously generated plasmid backbone containing three tandem repeats of TSs for miRNA-122 and genetically engineered TSs for miRNA-206.20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar miRNA-122 was previously described in several studies as liver specific,24Lagos-Quintana M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Identification of tissue-specific microRNAs from mouse.Curr. Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar,25Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. et al.A mammalian microRNA expression atlas based on small RNA library sequencing.Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3010) Google Scholar whereas utilization of the genetically engineered TSs for the muscle-specific miRNA-206 was shown to abrogate its cross-reactivity with miRNA-1 abundant in the heart.20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Thus, in our study, we have used two miRNA-122- and miRNA-206-controlled constructs: pdAAV-HO1-TS and control pdAAV-HO1-iTS with non-functional, inverted miRNA TS (iTS) region for generation of the scAAV9 vectors (scAAV9-HO1-TS and scAAV9-HO1-iTS, respectively). First, we evaluated the functionality of scAAV9 harboring the HO1-TS or HO1-iTS expression cassette in HEK293 cells, which are not only highly susceptible to AAV transduction but also do not express any of the regulatory miRNAs (Figure 1A). As a positive control, we used HO-1-carrying vector (scAAV9-HO1) devoid of any miRNA TSs (Figures 1B and 1C). To facilitate transgene detection, we transferred HEK293 cells for 24 h to hypoxic conditions (0.5% O2), because in human cells in hypoxia, endogenous HO-1 is downregulated by the Bach-1 repressor.26Kitamuro T. Takahashi K. Ogawa K. Udono-Fujimori R. Takeda K. Furuyama K. Nakayama M. Sun J. Fujita H. Hida W. et al.Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells.J. Biol. Chem. 2003; 278: 9125-9133Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Under hypoxic conditions, transgene overexpression from both vectors was clearly visible and comparable with control scAAV9-HO1 vector (Figures 1B and 1C), confirming the functionality of prepared constructs. In the next step, the vectors were tested in various murine cell lines corresponding to the tissues described as most efficiently transduced by systemically delivered AAV9.6Inagaki K. Fuess S. Storm T.A. Gibson G.A. Mctiernan C.F. Kay M.A. Nakai H. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8.Mol. Ther. 2006; 14: 45-53Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar We have chosen the AML-12 hepatocyte cell line expressing miRNA-122, C2C12 myoblasts that acquire expression of miRNA-206 as they differentiate to myotubes, and the HL-1 cardiomyocyte cell line characterized by lack of both of those miRNAs (Figure 1A). Because some of the cell lines (HL-1, AML12, and undifferentiated C2C12) turned out to be refractory to AAV9 transduction, they were subjected to transfection only with the corresponding plasmid. We observed considerably lower transgene expression in AML-12 cells transfected with pdAAV-HO1-TS, in comparison with the pdAAV-HO1-iTS with a non-functional regulatory region (Figure 1D). This effect was even more pronounced in differentiated C2C12 cells, where HO-1 expression from the scAAV9-HO1-TS vector was almost completely inhibited (Figure 1E). In contrast, in undifferentiated C2C12 myoblasts, in which the miRNA-206 was barely detectable (Figure 1A), the level of HO-1 protein did not significantly differ between miRNA-regulated pdAAV-HO1-TS and control pdAAV-HO1-iTS plasmid transfection (Figure 1F). Finally, we confirmed the functionality of the HO1-TS expression cassette in the transfected HL-1 cardiomyocyte cell line (Figure 1G) lacking both regulatory miRNAs (Figure 1A) and proceeded with the assessment of in vivo cardiac specificity of constructed vectors. The in vitro experiments demonstrated that AAV9 harboring miRNA-122 TSs and genetically engineered miRNA-206 TSs may provide heart-specific transduction. Thus, next, we systemically administered scAAV9-HO1-TS and scAAV9-HO1-iTS vectors to 3-month-old HO-1 KO female mice of the C57BL/6J × FVB strain. Then, after 4 weeks, the number of vector genome copies, transgene mRNA, and protein level in the heart, liver, and skeletal muscles were assessed. Genome copies (Figure 2A), as well as the HO-1 transcript (Figure 2B) and protein (Figure 2C), were detected in murine hearts and livers following scAAV9-HO1-iTS administration. In skeletal muscles, the background signal (untreated mice) was very high, and thus prevented the estimation of the number of AAV genome copies (Figure 2A); however, transcript and HO-1 protein were successfully detected (Figures 2B and 2C, respectively). What is more, in agreement with previously published data,20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar transgene expression from scAAV9-HO1-TS was completely repressed in the liver and skeletal muscles (Figure 2C), while AAV genomes and transgene mRNA (Figures 2A and 2B, respectively) were detected in those tissues, confirming successful transduction. Surprisingly, we observed a similar effect of scAAV9-HO1-TS in the heart, where despite the presence of AAV genomes (Figure 2A), no HO-1 protein was produced (Figure 2C). Thus, in order to explain this observation, we analyzed the expression of miRNA-122 in the hearts of these animals and found that miRNA-122, described so far as liver specific, is also expressed in the hearts of female C57BL/6J × FVB mice (Figure 2D, left panel, white bar). Still, its expression was much lower than in the liver (Figure 2D, right panel, white bar). Because sex differences may exist in miRNA profiles,27Guo L. Zhang Q. Ma X. Wang J. Liang T. miRNA and mRNA expression analysis reveals potential sex-biased miRNA expression.Sci. Rep. 2017; 7: 39812Crossref PubMed Scopus (74) Google Scholar we assessed the level of miRNA-122 also in the hearts of male mice of the C57BL/6J × FVB strain. It was considerably lower than in female mice (Figure 2D, left panel, black bar) and again much lower than in liver (Figure 2D, right panel, black bar), but still detectable. Of note, miRNA-122 expression in the heart was not dependent on HO-1, because the same results were obtained in wild-type animals (data not shown). In addition, we checked miRNA-206 expression because its presence in the heart may also cause the same off-target silencing; however, the level of this miRNA was below the detection limit (data not shown). Next, we wanted to verify whether a lower expression of miRNA-122 in the hearts of male C57BL/6J × FVB mice (Figure 2D) will result in a similar inhibition of transgene expression from the miRNA-controlled vector as in female mice. For this reason, we systemically administered scAAV9-HO1-TS and scAAV9-HO1-iTS vectors to 3-month-old HO-1 KO male mice of the C57BL/6J × FVB strain. We confirmed the presence of AAV genomes in hearts and livers (Figure 2E) and the presence of transgene mRNA (Figure 2F) in all analyzed tissues after delivery of both vectors. Moreover, even though in the hearts of male mice HO-1 protein was detectable after administration of scAAV9-HO1-TS vectors, we have noted a very potent (more than 10-fold) repression of transgene expression in comparison with the control scAAV9-HO1-iTS vector (Figure 2G). Previous studies utilized the miRNA-122-controlled vectors in different mouse strains than ours: male BALB/c,20Geisler A. Schön C. Größl T. Pinkert S. Stein E.A. Kurreck J. Vetter R. Fechner H. Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.Mol. Ther. 2013; 21: 924-933Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar male ICR,28Qiao C. Yuan Z. Li J. He B. Zheng H. Mayer C. Li J. Xiao X. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver.Gene Ther. 2011; 18: 403-410Crossref PubMed Scopus (103) Google Scholar or NMRI (information about sex was not provided).29Geisler A. Jungmann A. Kurreck J. Poller W. Katus H.A. Vetter R. Fechner H. Müller O.J. microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors.Gene Ther. 2011; 18: 199-209Crossref PubMed Scopus (89) Google Scholar Thus, we decided to investigate the miRNA-122 expression in the hearts of the mouse strains most widely used in the animal experimentation, namely, C57BL/6J, FVB, CBA, DBA, and BALB/c. In all cases, we used 3-month-old animals. miRNA-122 was clearly detected in both female and male mice of the FVB strain (Figure 3A), with significantly higher expression in females, similarly to our crossbred C57BL/6J × FVB mice (Figure 2D). In male BALB/c mice, miRNA-122 level was low but comparable with the level detected in male C57BL/6J × FVB mice (Figure 3B). Apart from that, we were able to detect miRNA-122 also in the hearts of male C57BL/6J, CBA, and DBA mouse strains, as well as in outbred animals (Figure 3B). Thus, strain-related variability of miRNA-122 level in mice was considerable. Next, we wanted to assess whether the data from murine hearts could reflect the individual differences in humans. For this purpose, we used heart tissue of patients suffering from various cardiomyopathies that was collected during cardiac transplantation. It turned out that miRNA-122 was detectable in all but one heart tissue, and its level differed between patients (Figure 3C). We also aimed to investigate the potential regulatory mechanisms underlying such considerable variability in miRNA-122 level in the heart. We focused on the description in the literature of the main transcriptional regulators of this miRNA expression in the liver, including hepatocyte nuclear factor (HNF) 1-alpha (HNF1α), HNF 3-alpha (HNF3α), HNF 3-beta (HNF3β), HNF 4-alpha (HNF4α), and retinoic X receptor-alpha (RXRα; RXRA).30Li Z.-Y. Xi Y. Zhu W.-N. Zeng C. Zhang Z.-Q. Guo Z.-C. Hao D.L. Liu G. Feng L. Chen H.Z. et al.Positive regulation of hepatic miR-122 expression by HNF4α.J. Hepatol. 2011; 55: 602-611Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar,31Coulouarn C. Factor V.M. Andersen J.B. Durkin M.E. Thorgeirsson S.S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties.Oncogene. 2009; 28: 3526-3536Crossref PubMed Scopus (597) Google Scholar Interestingly, the transcripts encoding all of these factors were present in the hearts of FVB, C57BL/6J, C57BL/6J × FVB, and BALB/c mice; however, the expression was uniform among investigated strains (data not shown). In the clinical samples, we detected greater variability in cardiac expression of HNF1α (Figure 3D), HNF3β (Figure 3E), and RXRA (Figure 3F). Interestingly, the profile of RXRA expression (Figure 3F) to some extent corresponded with miRNA-122 (Figure 3C), suggesting a possible regulatory mechanism of this miRNA expression in the human heart. Finally, we aimed to analyze whether transgene expression in human cardiomyocytes may be subjected to, similar as in animal studies, miRNA-122-mediated posttranscriptional regulation. Due to a well-known difficulty in obtaining primary human cardiomyocytes and their very limited proliferation potential,32Burridge P.W. Keller G. Gold J.D. Wu J.C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.Cell Stem Cell. 2012; 10: 16-28Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar,33Mercola M. Ruiz-Lozano P. Schneider M.D. Cardiac muscle regeneration: lessons from development.Genes Dev. 2011; 25: 299-309Crossref PubMed Scopus (137) Google Scholar for this purpose we utilized human iPSC-derived cardiomyocytes. First of all, we detected miRNA-122 in the cells from all three healthy donors (Figure 4A). Next, these iPSC-derived cardiomyocytes were transduced with scAAV9-GFP-TS and scAAV9-GFP-iTS. Here, the HO-1 harboring vectors were replaced with GFP-expressing ones to avoid a possible influence of endogenous HO-1 expression and its variability between different donors on the final outcome. Additionally, because cardiomyocytes were derived from different donors, we wanted to minimize the effect of potential variations in AAV9 receptor availability on the transduction efficiency. Thus, before transduction, cardiomyocytes were treated with neuraminidase to expose N-linked galactose on the surface of the cells, which is the primary receptor for AAV9.34Shen S. Bryant K.D. Brown S.M. Randell S.H. Asokan A. Terminal N-linked galactose is the primary receptor for adeno-associated virus 9.J. Biol. Chem. 2011; 286: 13532-13540Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar The level of miRNA-122 was relatively low in all three lines of human iPSC-derived cardiomyocytes (Figure 4A), but it was still sufficient to effectively inhibit transgene expression, because both the percentage of GFP+ cells (Figure 4B) and median fluorescence intensity (MFI) (Figure 4C) following scAAV9-GFP-TS were approximately by half smaller than after control scAAV9-GFP-iTS vector transduction. Despite numerous successful applications of gene therapy developed in recent years, many aspects regarding the design of safe and effective vectors remain challenging. One of these concerns" @default.
• W3024382227 created "2020-05-21" @default.
• W3024382227 creator A5018007407 @default.
• W3024382227 creator A5024795564 @default.
• W3024382227 creator A5039854767 @default.
• W3024382227 creator A5040188203 @default.
• W3024382227 creator A5051986223 @default.
• W3024382227 creator A5055691264 @default.
• W3024382227 creator A5068871940 @default.
• W3024382227 creator A5071331807 @default.
• W3024382227 creator A5082864592 @default.
• W3024382227 creator A5087034199 @default.
• W3024382227 date "2020-06-01" @default.
• W3024382227 modified "2023-10-16" @default.
• W3024382227 title "Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors" @default.
• W3024382227 cites W1517573369 @default.
• W3024382227 cites W1567904175 @default.
• W3024382227 cites W1907511822 @default.
• W3024382227 cites W1965306643 @default.
• W3024382227 cites W1971270809 @default.
• W3024382227 cites W1974997620 @default.
• W3024382227 cites W1996136504 @default.
• W3024382227 cites W2008289270 @default.
• W3024382227 cites W2017969617 @default.
• W3024382227 cites W2026531690 @default.
• W3024382227 cites W2028362572 @default.
• W3024382227 cites W2035146556 @default.
• W3024382227 cites W2042594259 @default.
• W3024382227 cites W2044474105 @default.
• W3024382227 cites W2048651210 @default.
• W3024382227 cites W2056490654 @default.
• W3024382227 cites W2058010019 @default.
• W3024382227 cites W2059073003 @default.
• W3024382227 cites W2061778565 @default.
• W3024382227 cites W2062298134 @default.
• W3024382227 cites W2065380345 @default.
• W3024382227 cites W2065566217 @default.
• W3024382227 cites W2071390582 @default.
• W3024382227 cites W2083599207 @default.
• W3024382227 cites W2086500311 @default.
• W3024382227 cites W2100923281 @default.
• W3024382227 cites W2102934400 @default.
• W3024382227 cites W2109136664 @default.
• W3024382227 cites W2116885047 @default.
• W3024382227 cites W2124929662 @default.
• W3024382227 cites W2139380535 @default.
• W3024382227 cites W2149146453 @default.
• W3024382227 cites W2150109976 @default.
• W3024382227 cites W2154578855 @default.
• W3024382227 cites W2168458927 @default.
• W3024382227 cites W2169219209 @default.
• W3024382227 cites W2171985637 @default.
• W3024382227 cites W2411838454 @default.
• W3024382227 cites W2412985586 @default.
• W3024382227 cites W2508549771 @default.
• W3024382227 cites W2567312126 @default.
• W3024382227 cites W2584921798 @default.
• W3024382227 cites W2617830918 @default.
• W3024382227 cites W2727482182 @default.
• W3024382227 cites W2741933191 @default.
• W3024382227 cites W2783365495 @default.
• W3024382227 cites W2805443401 @default.
• W3024382227 cites W2863150589 @default.
• W3024382227 cites W2896208536 @default.
• W3024382227 cites W2899943925 @default.
• W3024382227 cites W2912684492 @default.
• W3024382227 cites W2913212173 @default.
• W3024382227 cites W2971564040 @default.
• W3024382227 cites W3006887679 @default.
• W3024382227 cites W4250446838 @default.
• W3024382227 cites W787851930 @default.
• W3024382227 doi "https://doi.org/10.1016/j.omtm.2020.05.006" @default.
• W3024382227 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7270145" @default.
• W3024382227 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32518806" @default.
• W3024382227 hasPublicationYear "2020" @default.
• W3024382227 type Work @default.
• W3024382227 sameAs 3024382227 @default.
• W3024382227 citedByCount "10" @default.
• W3024382227 countsByYear W30243822272020 @default.
• W3024382227 countsByYear W30243822272021 @default.
• W3024382227 countsByYear W30243822272022 @default.
• W3024382227 countsByYear W30243822272023 @default.
• W3024382227 crossrefType "journal-article" @default.
• W3024382227 hasAuthorship W3024382227A5018007407 @default.
• W3024382227 hasAuthorship W3024382227A5024795564 @default.
• W3024382227 hasAuthorship W3024382227A5039854767 @default.
• W3024382227 hasAuthorship W3024382227A5040188203 @default.
• W3024382227 hasAuthorship W3024382227A5051986223 @default.
• W3024382227 hasAuthorship W3024382227A5055691264 @default.
• W3024382227 hasAuthorship W3024382227A5068871940 @default.
• W3024382227 hasAuthorship W3024382227A5071331807 @default.
• W3024382227 hasAuthorship W3024382227A5082864592 @default.
• W3024382227 hasAuthorship W3024382227A5087034199 @default.
• W3024382227 hasBestOaLocation W30243822271 @default.
• W3024382227 hasConcept C104317684 @default.
• W3024382227 hasConcept C145059251 @default.
• W3024382227 hasConcept C40767141 @default.
• W3024382227 hasConcept C53673613 @default.

• next