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1
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Chan T, Grisch-Chan HM, Schmierer P, Subotic U, Rimann N, Scherer T, Hetzel U, Bozza M, Harbottle R, Williams JA, Steblaj B, Ringer SK, Häberle J, Sidler X, Thöny B. Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection. Mol Ther Methods Clin Dev 2022; 24:268-279. [PMID: 35211639 PMCID: PMC8829443 DOI: 10.1016/j.omtm.2022.01.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Accepted: 01/16/2022] [Indexed: 11/09/2022]
Abstract
Hepatic gene therapy by delivering non-integrating therapeutic vectors in newborns remains challenging due to the risk of dilution and loss of efficacy in the growing liver. Previously we reported on hepatocyte transfection in piglets by intraportal injection of naked DNA vectors. Here, we established delivery of naked DNA vectors to target periportal hepatocytes in weaned pigs by hydrodynamic retrograde intrabiliary injection (HRII). The surgical procedure involved laparotomy and transient isolation of the liver. For vector delivery, a catheter was placed within the common bile duct by enterotomy. Under optimal conditions, no histological abnormalities were observed in liver tissue upon pressurized injections. The transfection of hepatocytes in all tested liver samples was observed with vectors expressing luciferase from a liver-specific promoter. However, vector copy number and luciferase expression were low compared to hydrodynamic intraportal injection. A 10-fold higher number of vector genomes and luciferase expression was observed in pigs using a non-integrating naked DNA vector with the potential for replication. In summary, the HRII application was less efficient (i.e., lower luciferase activity and vector copy numbers) than the intraportal delivery method but was significantly less distressful for the piglets and has the potential for injection (or re-injection) of vector DNA by endoscopic retrograde cholangiopancreatography.
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Affiliation(s)
- Tatjana Chan
- Department of Farm Animals, Division of Swine Medicine of the Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Hiu Man Grisch-Chan
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Philipp Schmierer
- Department of Small Animal Surgery, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Ulrike Subotic
- Department of Surgery, University Children's Hospital Basel, Basel, Switzerland
| | - Nicole Rimann
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Tanja Scherer
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Udo Hetzel
- Department of Pathology, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Matthias Bozza
- DNA Vector Laboratory, DKFZ Heidelberg, Heidelberg, Germany
| | | | | | - Barbara Steblaj
- Department of Diagnostics and Clinical Services, Section of Anesthesiology, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Simone K Ringer
- Department of Diagnostics and Clinical Services, Section of Anesthesiology, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Johannes Häberle
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Xaver Sidler
- Department of Farm Animals, Division of Swine Medicine of the Vetsuisse Faculty University of Zurich, Zurich, Switzerland
| | - Beat Thöny
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
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2
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Bellanti F, di Bello G, Iannelli G, Pannone G, Pedicillo MC, Boulter L, Lu WY, Tamborra R, Villani R, Vendemiale G, Forbes SJ, Serviddio G. Inhibition of nuclear factor (erythroid-derived 2)-like 2 promotes hepatic progenitor cell activation and differentiation. NPJ Regen Med 2021; 6:28. [PMID: 34039998 PMCID: PMC8155039 DOI: 10.1038/s41536-021-00137-z] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2020] [Accepted: 04/28/2021] [Indexed: 02/08/2023] Open
Abstract
The stem cell ability to self-renew and lead regeneration relies on the balance of complex signals in their microenvironment. The identification of modulators of hepatic progenitor cell (HPC) activation is determinant for liver regeneration and may improve cell transplantation for end-stage liver disease. This investigation used different models to point out the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) as a key regulator of the HPC fate. We initially proved that in vivo models of biliary epithelial cells (BECs)/HPC activation show hepatic oxidative stress, which activates primary BECs/HPCs in vitro. NRF2 downregulation and silencing were associated with morphological, phenotypic, and functional modifications distinctive of differentiated cells. Furthermore, NRF2 activation in the biliary tract repressed the ductular reaction in injured liver. To definitely assess the importance of NRF2 in HPC biology, we applied a xenograft model by inhibiting NRF2 in the human derived HepaRG cell line and transplanting into SCID/beige mice administered with anti-Fas antibody to induce hepatocellular apoptosis; this resulted in effective human hepatocyte repopulation with reduced liver injury. To conclude, NRF2 inhibition leads to the activation and differentiation of liver progenitors. This redox-dependent transcription factor represents a potential target to regulate the commitment of undifferentiated hepatic progenitors into specific lineages.
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Affiliation(s)
- Francesco Bellanti
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.
| | - Giorgia di Bello
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
| | - Giuseppina Iannelli
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
| | - Giuseppe Pannone
- Anatomical Pathology Unit, Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy
| | - Maria Carmela Pedicillo
- Anatomical Pathology Unit, Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy
| | - Luke Boulter
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK
| | - Wei-Yu Lu
- Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston Birmingham, UK
| | - Rosanna Tamborra
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
| | - Rosanna Villani
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
| | - Gianluigi Vendemiale
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
| | - Stuart J Forbes
- MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| | - Gaetano Serviddio
- Centre for Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
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Sakamoto K, Khai NC, Wang Y, Irie R, Takamatsu H, Matsufuji H, Kosai KI. Heparin-binding epidermal growth factor-like growth factor and hepatocyte growth factor inhibit cholestatic liver injury in mice through different mechanisms. Int J Mol Med 2016; 38:1673-1682. [PMID: 27779646 PMCID: PMC5117744 DOI: 10.3892/ijmm.2016.2784] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 09/02/2016] [Indexed: 12/23/2022] Open
Abstract
In contrast to hepatocyte growth factor (HGF), the therapeutic potential and pathophysiologic roles of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver diseases remain relatively unknown. To address the lack of effective pharmacologic treatments for cholestatic liver injuries, as well as to clarify the biologic features of these growth factors, we explored the effects of HB-EGF and HGF in mice with cholestatic liver injury induced by bile duct ligation (BDL). The mice were assessed 3, 5 and/or 14 days after BDL (acute, subacute and/or chronic phases, respectively) and intravenous injection of adenoviral vector expressing LacZ (control), HB-EGF, HGF, or HB-EGF and HGF. HB-EGF, HGF, or a combination of the growth factors exerted potent antioncotic (antinecrotic), antiapoptotic, anticholestatic, and regenerative effects on hepatocytes in vivo, whereas no robust antiapoptotic or regenerative effects were detected in interlobular bile ducts. Based on serum transaminase levels, the acute protective effects of HB-EGF on hepatocytes were greater than those of HGF. On the other hand, liver fibrosis and cholestasis during the chronic phase were more potently inhibited by HGF compared with HB-EGF. Compared with either growth factor alone, combining HB-EGF and HGF produced greater anticholestatic and regenerative effects during the chronic phase. Taken together, these findings suggest that HB-EGF and HGF inhibited BDL-induced cholestatic liver injury, predominantly by exerting acute cytoprotective and chronic antifibrotic effects, respectively; combining the growth factors enhanced the anticholestatic effects and liver regeneration during the chronic phase. Our results contribute to a better understanding of the pathophysiologic roles of HB-EGF and HGF, as well as to the development of novel effective therapies for cholestatic liver injuries.
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Affiliation(s)
- Kouichi Sakamoto
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Ngin Cin Khai
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Yuqing Wang
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Rie Irie
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Hideo Takamatsu
- Department of Pediatric Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Hiroshi Matsufuji
- Department of Pediatric Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
| | - Ken-Ichiro Kosai
- Department of Gene Therapy and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan
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4
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Gao L, Xie L, Long X, Wang Z, He CY, Chen ZY, Zhang L, Nan X, Lei H, Liu X, Liu G, Lu J, Qiu B. Efficacy of MRI visible iron oxide nanoparticles in delivering minicircle DNA into liver via intrabiliary infusion. Biomaterials 2013; 34:3688-96. [DOI: 10.1016/j.biomaterials.2013.01.094] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2012] [Accepted: 01/26/2013] [Indexed: 11/15/2022]
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5
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Kawabata A, Baoum A, Ohta N, Jacquez S, Seo GM, Berkland C, Tamura M. Intratracheal administration of a nanoparticle-based therapy with the angiotensin II type 2 receptor gene attenuates lung cancer growth. Cancer Res 2012; 72:2057-67. [PMID: 22389453 DOI: 10.1158/0008-5472.can-11-3634] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Targeted gene delivery, transfection efficiency, and toxicity concerns remain a challenge for effective gene therapy. In this study, we dimerized the HIV-1 TAT peptide and formulated a nanoparticle vector (dTAT NP) to leverage the efficiency of this cell-penetrating strategy for tumor-targeted gene delivery in the setting of intratracheal administration. Expression efficiency for dTAT NP-encapsulated luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated in Lewis lung carcinoma (LLC) cells cultured in vitro or in vivo in orthotopic tumor grafts in syngeneic mice. In cell culture, dTAT NP was an effective pDNA transfection vector with negligible cytotoxicity. Transfection efficiency was further increased by addition of calcium and glucose to dTAT/pDNA NP. In orthotopic tumor grafts, immunohistochemical analysis confirmed that dTAT NP successfully delivered pDNA to the tumor, where it was expressed primarily in tumor cells along with the bronchial epithelium. Notably, gene expression in tumor tissues persisted at least 14 days after intratracheal administration. Moreover, bolus administration of dTAT NP-encapsulated AT2R or TNF-related apoptosis-inducing ligand (TRAIL) pDNA markedly attenuated tumor growth. Taken together, our findings offer a preclinical proof-of-concept for a novel gene delivery system that offers an effective intratracheal strategy for administering lung cancer gene therapy.
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Affiliation(s)
- Atsushi Kawabata
- Department of Anatomy & Physiology, Kansas State University College of Veterinary Medicine, Manhattan, Kansas 66506, USA
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6
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Dai H, Jiang X, Leong KW, Mao HQ. Transient depletion of kupffer cells leads to enhanced transgene expression in rat liver following retrograde intrabiliary infusion of plasmid DNA and DNA nanoparticles. Hum Gene Ther 2010; 22:873-8. [PMID: 21091274 DOI: 10.1089/hum.2010.146] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
In this report, we have demonstrated that by temporarily removing Kupffer cells (KCs), the transgene expression levels mediated by retrograde intrabiliary infusion (RII) of plasmid DNA, polyethylenimine-DNA, and chitosan nanoparticles were enhanced by 1,927-, 131-, and 23,450-fold, respectively, in comparison with the respective groups without KC removal. KC removal also led to significantly prolonged transgene expression in the liver that received all three carriers. This increased transgene expression was correlated with significantly reduced serum tumor necrosis factor-α level as an indicator for KC activation. These results suggest that KC activation is a significant contributing factor to the lowered transgene expression by polycation-DNA nanoparticles delivered by RII. More importantly, the combination of RII and transient removal of KCs may be adopted as an effective approach to achieving high and persistent transgene expression in the liver mediated by nonviral nanoparticles.
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Affiliation(s)
- Hui Dai
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA
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7
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Atta HM. Gene therapy for liver regeneration: experimental studies and prospects for clinical trials. World J Gastroenterol 2010; 16:4019-30. [PMID: 20731015 PMCID: PMC2928455 DOI: 10.3748/wjg.v16.i32.4019] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2009] [Revised: 03/03/2010] [Accepted: 03/10/2010] [Indexed: 02/06/2023] Open
Abstract
The liver is an exceptional organ, not only because of its unique anatomical and physiological characteristics, but also because of its unlimited regenerative capacity. Unfolding of the molecular mechanisms that govern liver regeneration has allowed researchers to exploit them to augment liver regeneration. Dramatic progress in the field, however, was made by the introduction of the powerful tool of gene therapy. Transfer of genetic materials, such as hepatocyte growth factor, using both viral and non-viral vectors has proved to be successful in augmenting liver regeneration in various animal models. For future clinical studies, ongoing research aims at eliminating toxicity of viral vectors and increasing transduction efficiency of non-viral vectors, which are the main drawbacks of these systems. Another goal of current research is to develop gene therapy that targets specific liver cells using receptors that are unique to and highly expressed by different liver cell types. The outcome of such investigations will, undoubtedly, pave the way for future successful clinical trials.
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8
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Baoum A, Xie SX, Fakhari A, Berkland C. "Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide. Pharm Res 2009; 26:2619-29. [PMID: 19789962 PMCID: PMC4127430 DOI: 10.1007/s11095-009-9976-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2009] [Accepted: 09/14/2009] [Indexed: 02/02/2023]
Abstract
PURPOSE Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. METHODS Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. RESULTS TAT/pDNA complexes were condensed into small, 50-100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these "soft" crosslinks could be competitively disrupted to facilitate DNA release. CONCLUSION Small and stable TAT-Ca/pDNA complexes were obtained via "soft" calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.
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Affiliation(s)
- Abdulgader Baoum
- Department of Pharmaceutical Chemistry, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA
| | - Sheng-Xue Xie
- Department of Pharmaceutical Chemistry, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA
| | - Amir Fakhari
- Department of Bioengineering, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA
| | - Cory Berkland
- Department of Pharmaceutical Chemistry, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA
- Department of Chemical and Petroleum Engineering, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA
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9
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Crettaz J, Berraondo P, Mauleón I, Ochoa-Callejero L, Ochoa L, Shankar V, Barajas M, van Rooijen N, Kochanek S, Qian C, Prieto J, Hernández-Alcoceba R, González-Aseguinolaza G. Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice. Hepatology 2006; 44:623-32. [PMID: 16941711 DOI: 10.1002/hep.21292] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.
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Affiliation(s)
- Julien Crettaz
- Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain
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10
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Khai NC, Takahashi T, Ushikoshi H, Nagano S, Yuge K, Esaki M, Kawai T, Goto K, Murofushi Y, Fujiwara T, Fujiwara H, Kosai KI. In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: a comparative study to HGF. J Hepatol 2006; 44:1046-54. [PMID: 16466829 DOI: 10.1016/j.jhep.2005.10.027] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2005] [Revised: 09/20/2005] [Accepted: 10/10/2005] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. METHODS Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. RESULTS Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. CONCLUSIONS HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.
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Affiliation(s)
- Ngin Cin Khai
- Department of Gene Therapy and Regenerative Medicine, Gifu University School of Medicine, Gifu University Graduate School of Medicine, Gifu, Japan
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11
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Nakayama M, Both GW, Banizs B, Tsuruta Y, Yamamoto S, Kawakami Y, Douglas JT, Tani K, Curiel DT, Glasgow JN. An adenovirus serotype 5 vector with fibers derived from ovine atadenovirus demonstrates CAR-independent tropism and unique biodistribution in mice. Virology 2006; 350:103-15. [PMID: 16516257 DOI: 10.1016/j.virol.2006.01.037] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2005] [Revised: 12/23/2005] [Accepted: 01/26/2006] [Indexed: 01/09/2023]
Abstract
Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.
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Affiliation(s)
- Masaharu Nakayama
- Division of Human Gene Therapy, Department of Medicine, University of Alabama at Birmingham, 901 19th Street South BMR2-572, Birmingham, AL 35294-2180, USA.
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12
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Glasgow JN, Everts M, Curiel DT. Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 2006; 13:830-44. [PMID: 16439993 PMCID: PMC1781516 DOI: 10.1038/sj.cgt.7700928] [Citation(s) in RCA: 101] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions.
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Affiliation(s)
- JN Glasgow
- Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, Birmingham, AL, USA
| | - M Everts
- Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, Birmingham, AL, USA
- Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - DT Curiel
- Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, Birmingham, AL, USA
- Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA
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13
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Tang DQ, Lu S, Sun YP, Rodrigues E, Chou W, Yang C, Cao LZ, Chang LJ, Yang LJ. Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors. J Transl Med 2006; 86:83-93. [PMID: 16294197 PMCID: PMC3417286 DOI: 10.1038/labinvest.3700368] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.
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Affiliation(s)
- Dong-Qi Tang
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0275, USA
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14
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Oberholzer C, Tschoeke SK, Bahjat K, LaFace D, Hutchins B, Clare-Salzler MJ, Moldawer LL, Oberholzer A. In vivo transduction of thymic dendritic cells with adenovirus and its potential use in acute inflammatory diseases. Scand J Immunol 2005; 61:309-15. [PMID: 15853912 DOI: 10.1111/j.1365-3083.2005.01574.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Dendritic cells (DC) represent a potential target for gene therapy. In their ability to process antigens and present them to T cells, DC have been allocated a unique role as initiators of the immune response in both the innate and acquired immunity. Recent in vitro studies have showed the feasibility of DC transduction with adenoviral recombinants. In cancer therapy, targeting of DC with adenovirus has been proved to be effective in inhibiting tumour growth, as well as in reducing the number of tumour metastases. The aim of our study is to evaluate the feasibility of in vivo transduction of DC in a murine lymphocyte-rich compartment (thymus) as a potential treatment for acute inflammatory diseases. Nearly 50% of the total thymic DC were transduced with a first-generation adenoviral construct following intrathymic injection, and post-transductional inflammation was neglectable. Transduction of thymic cells with adenoviral recombinants was able to induce the expression of an intracellular protein (beta-galactosidase, green fluorescent protein), as well as the secretion of human interleukin-10, within the local compartment. Furthermore, this induction of the latter significantly decreased thymic apoptosis in the applied model of acute bacterial peritonitis (cecal ligation and puncture).
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Affiliation(s)
- C Oberholzer
- Department of Trauma and Reconstructive Surgery, CHARITE- University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany.
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15
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Sandhu AP, Lam AMI, Fenske DB, Palmer LR, Johnston M, Cullis PR. Calcium enhances the transfection potency of stabilized plasmid–lipid particles. Anal Biochem 2005; 341:156-64. [PMID: 15866540 DOI: 10.1016/j.ab.2005.02.033] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2005] [Indexed: 11/22/2022]
Abstract
Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid-lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca2+ can enhance the transfection potency of SPLP. It is shown that Ca2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.
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Affiliation(s)
- Ammen P Sandhu
- Department of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
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16
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Oberholzer C, Oberholzer A, Tschoeke SK, Minter RM, Bahjat FR, LaFace D, Hutchins B, Moldawer LL. Influence of recombinant adenovirus on liver injury in endotoxicosis and its modulation by IL-10 expression. ACTA ACUST UNITED AC 2005. [PMID: 15588421 DOI: 10.1177/09680519040100060301] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Adenovirus-based gene therapy offers a unique opportunity to target gene expression to the liver by systemic delivery. However, systemic administration of a first generation adenoviral construct elicits an inflammatory response leading to TNF-alpha-dependent liver injury. The aim of this study was to evaluate whether the systemic administration of recombinant adenovirus exacerbates a subsequent TNF-alpha-dependent liver injury induced by D-galactosamine and lipopolysaccharide. Surprisingly, low-dose adenovirus administration (10(5) particles) protects, while high-dose adenovirus (10(10) particles) is associated with an exaggerated hepatic inflammatory response from a subsequent D-galactosamine and lipopolysaccharide challenge. This exacerbation is TNF-alpha dependent, since treatment with a TNF inhibitor fully protects against the liver injury. Moreover, intravenous administration of an adenoviral construct expressing the anti-inflammatory protein interleukin-10 reduces TNF-alpha appearance and attenuates the increased hepatocyte injury. Taken together, this report demonstrates potential additive effects of TNF-alpha responses induced by adenovirus and other inflammatory signals, and suggests that the response can be mitigated by relative adenovirus particle dose or by inhibitors, such as TNF-binding protein or interleukin 10.
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Affiliation(s)
- Caroline Oberholzer
- Department of Surgery, University of Florida College of Medicine, Gainesville, Florida 32610-0286, USA
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17
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Imai J, Katagiri H, Yamada T, Ishigaki Y, Ogihara T, Uno K, Hasegawa Y, Gao J, Ishihara H, Sasano H, Mizuguchi H, Asano T, Oka Y. Constitutively active PDX1 induced efficient insulin production in adult murine liver. Biochem Biophys Res Commun 2005; 326:402-9. [PMID: 15582592 DOI: 10.1016/j.bbrc.2004.11.047] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2004] [Indexed: 11/19/2022]
Abstract
To generate insulin-producing cells in the liver, recombinant adenovirus containing a constitutively active mutant of PDX1 (PDX1-VP16), designed to activate target genes without the need for protein partners, was prepared and administered intravenously to streptozotocin (STZ)-treated diabetic mice. The effects were compared with those of administering wild-type PDX1 (wt-PDX1) adenovirus. Administration of these adenoviruses at 2x10(8)pfu induced similar levels of PDX1 protein expression in the liver. While wt-PDX1 expression exerted small effects on blood glucose levels, treatment with PDX1-VP16 adenovirus efficiently induced insulin production in hepatocytes, resulting in reversal of STZ-induced hyperglycemia. The effects were sustained through day 40 when exogenous PDX1-VP16 protein expression was undetectable in the liver. Endogenous PDX1 protein came to be expressed in the liver, which is likely to be the mechanism underlying the sustained effects. On the other hand, albumin and transferrin expressions were observed in insulin-producing cells in the liver, suggesting preservation of hepatocytic functions. Thus, transient expression of an active mutant of PDX1 in the liver induced sustained PDX1 and insulin expressions without loss of hepatocytic function.
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Affiliation(s)
- Junta Imai
- Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Japan
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18
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van Etten B, Eggermont AMM, Ambagtsheer G, van Tiel ST, ten Hagen TLM. Impaired neutralising antibody formation and high transduction efficacy after isolated hepatic perfusion with adenoviral vectors. Br J Cancer 2004; 91:1610-3. [PMID: 15480435 PMCID: PMC2409916 DOI: 10.1038/sj.bjc.6602151] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Local adenoviral gene transfer can be performed by means of isolated hepatic perfusion (IHP). This methodology is a very effective and safe way to deliver adenoviral vectors. We studied the immune response after IHP. A decreased neutralising antibody formation was observed, offering possibilities for further research in the field of gene therapy in isolated perfusion settings.
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Affiliation(s)
- B van Etten
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands
| | - A M M Eggermont
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands. E-mail:
| | - G Ambagtsheer
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands
| | - S T van Tiel
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands
| | - T L M ten Hagen
- Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Department of Surgical Oncology, PO Box 5201, 3008 AE Rotterdam, The Netherlands
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Shayakhmetov DM, Li ZY, Gaggar A, Gharwan H, Ternovoi V, Sandig V, Lieber A. Genome size and structure determine efficiency of postinternalization steps and gene transfer of capsid-modified adenovirus vectors in a cell-type-specific manner. J Virol 2004; 78:10009-22. [PMID: 15331734 PMCID: PMC514985 DOI: 10.1128/jvi.78.18.10009-10022.2004] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.
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Affiliation(s)
- Dmitry M Shayakhmetov
- Division of Medical Genetics, Box 357720, University of Washington, Seattle, WA 98195, USA
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20
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Pietersen AM, Rutjes SA, van Tongeren J, Vogels R, Wesseling JG, Noteborn MHM. The tumor-selective viral protein apoptin effectively kills human biliary tract cancer cells. J Mol Med (Berl) 2003; 82:56-63. [PMID: 14647920 DOI: 10.1007/s00109-003-0486-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2003] [Accepted: 08/13/2003] [Indexed: 02/06/2023]
Abstract
Biliary tract cancer, or cholangiocarcinoma, has a poor prognosis. Resection is the only curative treatment, but only a minority of patients are eligible. Chemotherapy and gamma-irradiation are merely palliative, as they are unable to remove the malignancy completely. The chicken anemia virus-derived protein apoptin induces apoptosis in a wide range of human tumor cells and is not hindered by mutations inactivating p53 or by overexpression of Bcl-2, changes known to frustrate chemotherapy and radiation therapy. We examined whether apoptin kills human biliary tract cancer cells. Expression of apoptin by means of plasmids caused extensive cell death in three independent cholangiocarcinoma cell lines, CC-LP, CC-SW, and Mz-ChA-1, regardless of their oncogenic mutations, which included inactivated p16 and p53 and the disruption of the transforming growth factor beta signaling pathway. In vitro delivery of apoptin by an adenoviral vector completely eradicated cholangiocarcinoma cells. Moreover, coexpression of the broad-spectrum caspase inhibitor p35 with apoptin only delayed the induced cell death. Changes in nuclear morphology still occurred early after transfection, and nuclei eventually disintegrated, suggesting that apoptin-induced cell death in these cells is not blocked by mutations in either the initiation or execution phase of apoptosis. The efficient induction of cell death by apoptin in cholangiocarcinoma cell lines makes apoptin an attractive candidate for molecular therapy of biliary tract cancer.
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Affiliation(s)
- Alexandra M Pietersen
- Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden, The Netherlands
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21
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Barton KN, Tyson D, Stricker H, Lew YS, Heisey G, Koul S, de la Zerda A, Yin FF, Yan H, Nagaraja TN, Randall KA, Jin GK, Fenstermacher JD, Jhiang S, Ho Kim J, Freytag SO, Brown SL. GENIS: gene expression of sodium iodide symporter for noninvasive imaging of gene therapy vectors and quantification of gene expression in vivo. Mol Ther 2003; 8:508-18. [PMID: 12946325 DOI: 10.1016/s1525-0016(03)00153-9] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase (mutTK(SR39)) fusion gene and the hNIS gene was developed. Ad5-yCD/mutTK(SR39)rep-hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na(99m)TcO(4) using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, (99m)TcO(4)(-) uptake was readily detected in the prostate using viral dose levels (10(10) to 10(12) viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of (99m)TcO(4)(-) uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.
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22
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Ber I, Shternhall K, Perl S, Ohanuna Z, Goldberg I, Barshack I, Benvenisti-Zarum L, Meivar-Levy I, Ferber S. Functional, persistent, and extended liver to pancreas transdifferentiation. J Biol Chem 2003; 278:31950-7. [PMID: 12775714 DOI: 10.1074/jbc.m303127200] [Citation(s) in RCA: 220] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Pancreatic and duodenal homeobox gene-1 (PDX-1) regulates pancreas development during embryogenesis, whereas in the adult it controls beta-cell function. Here we analyze whether PDX-1 functions as a pancreatic differentiation factor and a bona fide master regulator when ectopically expressed in mature fully differentiated liver in vivo. By ectopic and transient PDX-1 expression in liver in vivo, using the first generation recombinant adenoviruses, we demonstrate that PDX-1 induces in liver a wide repertoire of both exocrine and endocrine pancreatic gene expression. Moreover, PDX-1 induces its own expression (auto-induction), which in turn may explain the long lasting nature of the "liver to pancreas" transdifferentiation. Insulin as well glucagon-producing cells are mainly located in the proximity of hepatic central veins, possibly allowing direct hormone release into the bloodstream, without affecting normal hepatic function. Importantly, we demonstrate that hepatic insulin production triggered by Ad-CMV-PDX-1 recombinant adenovirus administration is functional and prevents streptozotocin-induced hyperglycemia in Balb/c mice even 8 months after the initial treatment. We conclude that PDX-1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also from mature liver. Transconversion of liver to pancreas may serve as a novel approach for generating endocrine-pancreatic tissue that can replace malfunctioning beta-cells in diabetics.
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Affiliation(s)
- Idit Ber
- Endocrine Institute, Sheba Medical Center, Tel-Hashomer 52621, Israel
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23
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Varnavski AN, Schlienger K, Bergelson JM, Gao GP, Wilson JM. Efficient transduction of human monocyte-derived dendritic cells by chimpanzee-derived adenoviral vector. Hum Gene Ther 2003; 14:533-44. [PMID: 12718764 DOI: 10.1089/104303403764539323] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Using recombinant adenoviruses (Ads) to target host dendritic cells (DCs) presents an attractive prospect for immunization. The efficacy of commonly used human Ad-derived gene transfer vectors for antigen delivery in humans is often compromised by preexisting anti-Ad immunity, acquired by the majority of human population as a result of frequent naturally occurring virus infections. As an alternative vector we propose chimpanzee-derived recombinant adenoviruses, which are poorly neutralized by human sera. In the present study we examine the ability of one such vector, AdC68, to transduce and activate human monocyte-derived DCs in culture. We found that AdC68 could efficiently transduce both immature and mature DCs at levels similar to those by the human serotype 5 Ad recombinant. Exposure of immature DCs to AdC68 did not alter the expression of activation and maturation marker molecules on the cell surface. Nevertheless, the transduction induced DCs to secrete interferon alpha and interleukin (IL)-6, but not IL-12 or tumor necrosis factor alpha. In addition, AdC68-transduced immature DCs could stimulate proliferation of autologous T lymphocytes. This is the first report describing a chimpanzee-derived recombinant Ad as a vector for transduction of human DCs.
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Affiliation(s)
- Andrei N Varnavski
- Department of Medicine, Medical Genetics Division, University of Pennsylvania School of Medicine, Philadelphia 19104, USA
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24
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Iwaki T, Sugimura M, Nishihira J, Matsuura T, Kobayashi T, Kanayama N. Recombinant adenovirus vector bearing antisense macrophage migration inhibitory factor cDNA prevents acute lipopolysaccharide-induced liver failure in mice. J Transl Med 2003; 83:561-70. [PMID: 12695559 DOI: 10.1097/01.lab.0000062857.26210.ef] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in delayed hypersensitivity and cellular immunity. MIF also acts as a proinflammatory cytokine and counterregulates the anti-inflammatory effects of glucocorticoids. Exogenous gene transfer mediated by adenovirus is useful to study a particular molecular function as well as to develop gene therapy strategies. A recombinant adenovirus containing sense and antisense murine MIF (mMIF) cDNA inserts was constructed using a cosmid-terminal protein complex method. The sense mMIF adenovirus (AxCA-mMIFS) efficiently induced mMIF in COS-7 cells that endogenously lack mMIF in a dose-dependent manner. In contrast, the antisense mMIF adenovirus (AxCA-mMIFAS) inhibited the expression of mMIF in NIH3T3 cells in a dose-dependent manner. To assess the pathophysiologic role of MIF in acute liver failure, we induced acute onset of liver damage in mice (male Jcl:ICR) by a combined treatment of Bacille Calmette-Guerin (BCG) and lipopolysaccharide (LPS). mMIF level in the liver of mice infected with AxCA-mMIFAS showed a significant reduction in MIF production in response to BCG-LPS compared with mice treated without viral infection and with AxCA-mMIFS. In addition, the immunohistochemical staining demonstrated that F4/80 antigen on macrophage was enhanced in liver infected with AxCA-mMIFS but reduced in liver infected with AxCA-mMIFAS. The staining intensity is correlated with the mMIF antigen level in liver tissue. The survival rate of mice infected with AxCA-mMIFAS was significantly higher than that of mice treated with PBS and infected with AxCA-LacZ in BCG-LPS. These results suggest that inhibition of MIF production, using recombinant adenovirus bearing the antisense MIF gene, reduced the mortality rate in BCG-LPS-induced liver failure in mice. This finding might aid in the further development of gene therapy targeting MIF.
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Affiliation(s)
- Takayuki Iwaki
- Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan
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25
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Sze DY, Freeman SM, Slonim SM, Samuels SL, Andrews JC, Hicks M, Ahrar K, Gupta S, Reid TR. Dr. Gary J. Becker Young Investigator Award: intraarterial adenovirus for metastatic gastrointestinal cancer: activity, radiographic response, and survival. J Vasc Interv Radiol 2003; 14:279-90. [PMID: 12631632 DOI: 10.1097/01.rvi.0000058422.01661.1e] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
PURPOSE To determine the antitumoral activity and radiographic response pattern of intraarterial administration of a selective replication-competent adenovirus in patients with hepatic metastases from gastrointestinal carcinomas. MATERIALS AND METHODS Thirty-five patients were treated, seven in the dose-escalation phase and 28 at high doses. Inclusion criteria allowed mild laboratory value and performance status abnormalities and as much as 50% replacement of hepatic volume by tumor. An attenuated adenovirus that selectively replicates in p53-deficient cells (Onyx-015) was administered by hepatic arterial infusion at doses as high as 2 x 10(12) particles for two cycles. Subsequent cycles (maximum of eight total) were administered in combination with intravenous 5-fluorouracil (5-FU) and leucovorin. RESULTS Tumor responses were demonstrated in combination with chemotherapy, even in 5-FU-resistant patients. The 15 patients who responded radiographically showed a pattern of acute tumor enlargement despite normalization of laboratory and clinical parameters, followed by very slow regression of tumor size. Radiographic response did not correlate with p53 status. Median survival of radiographic responders (475 days) was significantly longer than that of nonresponders (143 days). CONCLUSIONS Hepatic arterial infusion of the replication-selective adenovirus Onyx-015 in combination with chemotherapy resulted in tumor regressions in select patients, including some in whom previous chemotherapy had failed. A biphasic radiographic response pattern was demonstrated. The mechanism of action appears to be more complex than that seen in vitro.
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Affiliation(s)
- Daniel Y Sze
- Division of Cardiovascular and Interventional Radiology, Stanford University Medical Center, Stanford, CA 94305-5642, USA.
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26
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Ehrhardt A, Peng PD, Xu H, Meuse L, Kay MA. Optimization of cis-acting elements for gene expression from nonviral vectors in vivo. Hum Gene Ther 2003; 14:215-25. [PMID: 12639302 DOI: 10.1089/10430340360535779] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
While naked DNA gene transfer in vivo usually results in transient gene expression, in some cases long-term transgene expression can be achieved. Here we demonstrate that cis-acting DNA elements flanking the transgene expression cassette and components in the plasmid backbone can significantly influence expression levels from nonviral vectors. To demonstrate this, we administered our most robust human coagulation factor IX (hFIX) expression cassette placed in two different plasmid backbones, into the livers of mice, by hydrodynamic transfection. We found that placing the expression cassette within a minimal plasmid vector pHM5, a modified version of pUC19, resulted in 10 times higher serum hFIX expression levels (up to 20000 ng/ml, 400% of normal hFIX serum levels), compared to a pBluescript backbone. To optimally increase expression levels from a nonviral vector, we added matrix attachment regions (MARs) as cis-acting DNA elements flanking the hFIX expression cassette. We detected five fold higher hFIX expression levels in vivo for up to 1-year posttransfection from a vector that contained the chicken MAR from the lysozyme locus. Together, the present work demonstrates that in addition to the transgene expression cassette, cis-acting DNA elements within and outside of the plasmid backbone need to be evaluated to achieve optimal expression levels in a nonviral gene therapy approach.
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Affiliation(s)
- Anja Ehrhardt
- Departments of Pediatrics and Genetics, School of Medicine, Stanford University, Stanford, CA 94305, USA
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27
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Varnavski AN, Zhang Y, Schnell M, Tazelaar J, Louboutin JP, Yu QC, Bagg A, Gao GP, Wilson JM. Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity. J Virol 2002; 76:5711-9. [PMID: 11991999 PMCID: PMC137042 DOI: 10.1128/jvi.76.11.5711-5719.2002] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.
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Affiliation(s)
- Andrei N Varnavski
- Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania and The Wistar Institute, Philadelphia, Pennsylvania 19104, USA
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28
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Ehrhardt A, Kay MA. A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood 2002; 99:3923-30. [PMID: 12010790 DOI: 10.1182/blood.v99.11.3923] [Citation(s) in RCA: 103] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus-treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 x 10(9) transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor-alpha serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that the cis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.
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Affiliation(s)
- Anja Ehrhardt
- Department of Pediatrics and Genetics, School of Medicine, Stanford University, CA 94305, USA
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29
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McKay T, Reynolds P, Jezzard S, Curiel D, Coutelle C. Secretin-mediated gene delivery, a specific targeting mechanism with potential for treatment of biliary and pancreatic disease in cystic fibrosis. Mol Ther 2002; 5:447-54. [PMID: 11945072 DOI: 10.1006/mthe.2002.0560] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Gene therapy directed to the gastroenterological manifestations of cystic fibrosis (CF) would ideally be administered systemically. Such delivery would require efficient targeting at the cellular level to achieve a safe and effective therapy. Here we describe gene delivery using the secretin receptor (SR) as a basolateral target specific to the biliary and pancreatic epithelia affected in CF patients. We describe here targeting of a polycation-based nonviral gene delivery vector and retargeting of an adenoviral vector to cells expressing the SR in vitro. We were able to transfect cells expressing the SR up to 10-fold more efficiently than those not expressing the SR with a targeted polycation, SecGGC-lPEI. This targeting effect was secretin-specific and substantially reduced by competing secretin. SR-retargeted adenovirus transduced SR-expressing cells at more than sixfold higher levels than adenovirus alone. The SR may be an effective target for targeting systemically applied viral and nonviral gene delivery constructs to disease-affected tissues in CF patients.
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Affiliation(s)
- Tristan McKay
- Molecular Genetics, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College School of Medicine, South Kensington, London, SW7 2AZ, UK.
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30
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Li XK, Kosuga M, Tokieda K, Kanaji A, Fukuhara Y, Hashimoto M, Okabe K, Yaginuma H, Yamada M, Suzuki S, Okuyama T. Prolongation of transgene expression by coexpression of cytokine response modifier a in rodent liver after adenoviral gene transfer. Mol Ther 2002; 5:262-8. [PMID: 11863415 DOI: 10.1006/mthe.2002.0543] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The short duration of expression of the transgenes is a major barrier to the clinical application of adenovirus-mediated gene therapy for hepatic enzyme deficiencies. Previous reports show that Fas-mediated apoptosis has a pivotal role in the rapid elimination of adenovirus-infected hepatocytes. After considering this result and our recent observation that murine hepatocytes can be protected from Fas-mediated apoptosis by expressing cytokine response modifier A (CrmA) in vivo, we hypothesized that CrmA coexpression could also prevent adenovirus-infected hepatocytes from rapid elimination and that this would make prolonged transgene expression achievable in vivo. To examine this, mice with congenital deficiency of lysosomal beta-glucuronidase (GUSB) were infected with recombinant adenoviruses expressing both CrmA and GUSB, and the duration of transgene expression was evaluated. The serum GUSB activity in the mice injected with a recombinant adenovirus expressing GUSB only became undetectable 60 days after the injection, whereas higher than normal GUSB activity was observed for at least 120 days in mice injected with adenoviruses expressing both GUSB and CrmA. Furthermore, we showed that exogenous CrmA expression could prevent the adenovirus-infected hepatocytes from cell death induced by cytotoxic T lymphocytes in vitro. These observations indicate that transgene expression after administration of E1-deleted adenovirus is prolonged by coexpression of the antiapoptotic protein CrmA.
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Affiliation(s)
- Xiao-Kang Li
- Department of Experimental Surgery, National Children's Medical Research Center, Tokyo 154-8509, Japan
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31
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Sedlacek HH. Pharmacological aspects of targeting cancer gene therapy to endothelial cells. Crit Rev Oncol Hematol 2001; 37:169-215. [PMID: 11248576 DOI: 10.1016/s1040-8428(00)00113-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Targeting cancer gene therapy to endothelial cells seems to be a rational approach, because (a) a clear correlation exists between proliferation of tumor vessels and tumor growth and malignancy, (b) differences of cell membrane structures between tumor endothelial cells and normal endothelial cells exist which could be used for targeting of vectors and (c) tumor endothelial cells are accessible to vector vehicles in spite of the peculiarities of the transvascular and interstitial blood flow in tumors. Based on the knowledge on the pharmacokinetics of macromolecules it can be concluded that vectors targeting tumor endothelial cells should own a long blood residence time after intravascular application. This precondition seems to be fulfilled best by vectors exhibiting a slight anionic charge. A long blood residence time would allow the formation of a high amount of complexes between tumor endothelial cells and vector particles. Such high amount of complexes should enable a high transfection rate of tumor endothelial cells. In view of their pharmacokinetic behavior nonviral vectors seem to be more suitable for in vivo targeting tumor endothelial cells than viral vectors. Specific binding of nonviral vectors to tumor endothelial cells should be enhanced by multifunctional ligands and the transduction efficiency should be improved by cationic carriers. Effector genes should encode proteins potent enough to induce reactions which eliminate the tumor tissue. To be effective to that degree such proteins should induce self-amplifying antitumor reactions. Examples for proteins which have the potential to induce such self-amplifying tumor reactions are proteins endowed with antiangiogenic and antiproliferative activity, enzymes which convert prodrugs into drugs and possibly also proteins which induce embolization of tumor vessels. The pharmacological data for such examples are discussed in detail.
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Affiliation(s)
- H H Sedlacek
- Aventis Pharma Deutschland GmbH, Central Biotechnology, PO Box 1140, 35001, Marburg, Germany.
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Westgren M, Shields LE. In utero stem cell transplantation in humans. ERNST SCHERING RESEARCH FOUNDATION WORKSHOP 2001:197-221. [PMID: 11105262 DOI: 10.1007/978-3-662-04469-8_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/15/2023]
Affiliation(s)
- M Westgren
- Department of Obstetrics and Gynecology, Huddinge University Hospital, Sweden
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33
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Roehl HH, Leibbrandt ME, Greengard JS, Kamantigue E, Glass WG, Giedlin M, Boekelheide K, Johnson DE, Jolly DJ, Sajjadi NC. Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction. Hum Gene Ther 2000; 11:2529-40. [PMID: 11119423 DOI: 10.1089/10430340050208000] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.
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Affiliation(s)
- H H Roehl
- Chiron Corporation, Center for Gene Therapy, San Diego, CA 92121, USA.
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Shields LE, Kiem HP, Andrews RG. Highly efficient gene transfer into preterm CD34 hematopoietic progenitor cells. Am J Obstet Gynecol 2000; 183:732-7. [PMID: 10992201 DOI: 10.1067/mob.2000.106752] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
OBJECTIVE Retrovirus-mediated gene transfer has been shown to transduce CD34(+) cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks' gestation) and term (37-41 weeks' gestation) umbilical cord blood CD34(+) hematopoietic progenitor cells. STUDY DESIGN CD34(+) cells were purified from cyropreserved early (23-28 weeks' gestation) and term (37-40 weeks' gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34(+) cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus-containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. RESULTS Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34(+) hematopoietic progenitor cells from early (58.4% +/- 11.8%) and term (63.2% +/- 12.5%) gestation fetal umbilical cord blood. CONCLUSION CD34(+) hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34(+) and CD38(-) cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed.
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Affiliation(s)
- L E Shields
- Division of Perinatal Medicine, Department of Obstetrics and Gynecology, University of Washington School of School of Medicine, Seattle, WA 98195-6460, USA
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35
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Yang CQ, Wang JY, Fang GT, Liu JJ, Guo JS. Comparison between intravenous and peritoneal route on liver targeted uptake and expression of plasmid delivered by Glyco-poly-L-lysine. World J Gastroenterol 2000; 6:508-512. [PMID: 11819638 PMCID: PMC4723548 DOI: 10.3748/wjg.v6.i4.508] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To compare the effects of intravenous route and peritoneal route on liver targeted uptake and expression of plasmid delivered by galactose-terminal glyco-poly-L-lysine (G-PLL).
METHODS: The plasmid pTM/MMP-1 which could be expressed in eukaryotic cells was bound to G-PLL, and was then transferred into Wistar rats by intra venous and intraperitoneal injection. The expression and distribution of the plasmid were observed at different time periods by in situ hybridization and im munohistochemistry.
RESULTS: The plasmid could be expressed significantly within 24 h a fter being transferred in vivo by both intravenous and intraperitoneal routes. One week later the expression began to decrease, and could still be observed three weeks later. Although both the intravenous and intraperitoneal route could target-specifically deliver the plasmid to the liver, the effect of the former was better as compared to that of the latter.
CONCLUSION: Intravenous route is better for liver targeted uptake and expression of G-PLL-bound plasmids than the peritoneal route.
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Wiener SM, Hoyt RF, Deleonardis JR, Clevenger RR, Jeffries KR, Nagashima K, Mandel M, Owens J, Eckhaus M, Lutz RJ, Safer B. Manometric changes during retrograde biliary infusion in mice. Am J Physiol Gastrointest Liver Physiol 2000; 279:G49-66. [PMID: 10898746 DOI: 10.1152/ajpgi.2000.279.1.g49] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [(14)C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 A in diameter to specific histological compartments.
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Affiliation(s)
- S M Wiener
- Molecular Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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37
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Ueki K, Yamauchi T, Tamemoto H, Tobe K, Yamamoto-Honda R, Kaburagi Y, Akanuma Y, Yazaki Y, Aizawa S, Nagai R, Kadowaki T. Restored insulin-sensitivity in IRS-1-deficient mice treated by adenovirus-mediated gene therapy. J Clin Invest 2000; 105:1437-45. [PMID: 10811851 PMCID: PMC315460 DOI: 10.1172/jci7656] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Deltap85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin's metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Deltap85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Deltap85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.
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Affiliation(s)
- K Ueki
- Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
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Printz MA, Gonzalez AM, Cunningham M, Gu DL, Ong M, Pierce GF, Aukerman SL. Fibroblast growth factor 2-retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors. Hum Gene Ther 2000; 11:191-204. [PMID: 10646650 DOI: 10.1089/10430340050016265] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Targeted vectors provide a number of advantages for systemic and local gene delivery strategies. Several groups have investigated the utility of using various ligands to alter the tropism of adenovirus (Ad) vectors. We have previously demonstrated that fibroblast growth factor (FGF) ligands can specifically target DNA transfection and Ad transduction through high-affinity FGF receptors (FGFRs). FGFRs are overexpressed in abnormally proliferating tissues, such as malignancies. The present studies explore the effects of retargeting with FGF2 on the tissue localization pattern and the systemic toxicity of Ad in mice. Results of semiquantitative PCR analyses indicate that the distribution of FGF2-Ad vector genome sequences after intravenous administration in mice is altered. Markedly lower amounts (10- to 20-fold) of FGF2-Ad localize to the liver when compared with native Ad. This decrease in liver deposition translates into a significant reduction in subsequent toxicity as measured by serum transaminases and histopathology in mice injected with FGF2-AdHSV-thymidine kinase with and without ganciclovir administration. In an intraperitoneal model of ovarian cancer, FGF2-Ad generates increased transgene expression in tumor tissue when compared with Ad. Taken together, these results indicate that the retargeting of Ad with FGF2 results in a more efficient vector system for systemic and regional gene therapy applications, with concomitant lower levels of systemic toxicity.
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Affiliation(s)
- M A Printz
- Selective Genetics, Inc., San Diego, CA 92121, USA.
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Hofmann C, Löser P, Cichon G, Arnold W, Both GW, Strauss M. Ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo. J Virol 1999; 73:6930-6. [PMID: 10400791 PMCID: PMC112778 DOI: 10.1128/jvi.73.8.6930-6936.1999] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contained the cDNA of the human alpha1-antitrypsin (hAAT) gene linked to the Rous sarcoma virus promoter was generated and administered systemically to mice. The level and duration of hAAT gene expression was similar to that achieved with an hAd counterpart in both immunocompetent and immunodeficient mice. However, the tissue distribution of the OAV vector differed from that observed for hAd vectors in that the liver was not the dominant target. Significantly, we demonstrated efficient gene transfer with the OAV vector into mice immunized with hAd vectors and vice versa. We also confirm that the immune response to a transgene product can prevent its functional expression following sequential application of a vector. Our results suggest a possible solution to endemic humoral immunity against currently used hAd vectors and should therefore have an impact on the design of improved gene therapy protocols utilizing adenovirus vectors.
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Affiliation(s)
- C Hofmann
- HepaVec AG für Gentherapie, 13122 Berlin-Buch, Germany.
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Bilbao G, Contreras JL, Eckhoff DE, Mikheeva G, Krasnykh V, Douglas JT, Thomas FT, Thomas JM, Curiel DT. Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 1999; 230:185-93. [PMID: 10450732 PMCID: PMC1420860 DOI: 10.1097/00000658-199908000-00008] [Citation(s) in RCA: 116] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
OBJECTIVE To examine the possibility of reducing ischemia-reperfusion injury (I/R injury) to the mouse liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic human Bcl-2 gene. SUMMARY BACKGROUND DATA Ischemia-reperfusion injury has been demonstrated in a number of clinically relevant diseases such as myocardial infarction, cerebrovascular disease, sepsis, peripheral vascular disease, and organ transplantation. In this regard, apoptosis plays a central role. METHODS Normal C57BL/6 mice were used. An adenovirus (deltaE1) vector containing the human Bcl-2 gene was developed in the authors' laboratory. An adenovirus vector encoding an irrelevant gene (beta-galactosidase, AdCMVLacZ) was used as a control. Taking advantage of the hepatotropic properties of adenovirus vectors, gene transfer was performed with 1 x 10(9) plaque-forming units by intravenous tail injection, 48 hours before the ischemic injury. Ischemic-reperfusion injury was induced by temporal and segmental occlusion of hepatic blood flow. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activity was measured using standard assays. Liver biopsies were obtained before and 6 hours after I/R injury for morphologic assessment, and apoptosis was determined in situ with a histochemical assay. RESULTS The expression of AdCMVhBcl-2 vector was confirmed by reverse transcription-polymerase chain reaction and functionally validated in apoptotic studies in endothelial cells. Expression of the Bcl-2 gene protects against I/R injury, as shown by a significant decrease in transaminases (p < 0.05) and necrosis and apoptosis (p < 0.001), and permanent survival (p < 0.0001), compared with sham-operated animals and animals treated with AdCMVLacZ. CONCLUSIONS Genetic modification of the liver to induce cytoprotection has potential applications to prevent I/R injury to the liver in surgical interventions, including liver transplantation.
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Affiliation(s)
- G Bilbao
- Gene Therapy Program, Department of Surgery, University of Alabama at Birmingham, 35294, USA
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Bilbao G, Contreras JL, Zhang HG, Pike MJ, Overturf K, Mikheeva G, Krasnykh V, Curiel DT. Adenovirus-mediated gene expression in vivo is enhanced by the antiapoptotic bcl-2 gene. J Virol 1999; 73:6992-7000. [PMID: 10400798 PMCID: PMC112785 DOI: 10.1128/jvi.73.8.6992-7000.1999] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.
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Affiliation(s)
- G Bilbao
- Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
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42
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Ziegler RJ, Yew NS, Li C, Cherry M, Berthelette P, Romanczuk H, Ioannou YA, Zeidner KM, Desnick RJ, Cheng SH. Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer. Hum Gene Ther 1999; 10:1667-82. [PMID: 10428212 DOI: 10.1089/10430349950017671] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.
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Affiliation(s)
- R J Ziegler
- Genzyme Corporation, Framingham, MA 01701-9322, USA
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Kurata H, Liu CB, Valkova J, Koch AE, Yssel H, Hirabayashi Y, Inoue T, Yokota T, Arai K. Recombinant adenovirus vectors for cytokine gene therapy in mice. J Allergy Clin Immunol 1999; 103:S471-84. [PMID: 10329851 DOI: 10.1016/s0091-6749(99)70164-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
BACKGROUND Adenoviruses have several specific features useful for gene therapy. They infect various lineages of cells irrespective of cell cycle status. However, the exact mechanism of their infection and in vivo kinetics as a gene expression vector have not been elucidated. OBJECTIVE Using adenovirus vectors expressing marker genes, we examined the infectivity of these vectors (including cellular and tissue tropism), the duration and intensity of transgene expression, and the side effects. METHODS Various cells were infected with adenovirus expressing LacZ gene at various doses, and beta-galactosidase activity was measured and compared in relation with dose, time course, and cellular vitronectin receptor. Mice were injected with adenoviruses expressing LacZ, luciferase and GM-CSF, and in vivo gene expression was examined. RESULTS Adenovirus infection induced viral dose-dependent transgene expression that persisted for 2 weeks. Adherent cells were infected much more efficiently than nonadherent cells, probably because the former expressed much higher levels of the vitronectin receptor, one of the main receptors for adenovirus. Studies performed in mice with luciferase-expressing adenovirus revealed that the liver was the main target organ after intravenous injection and showed that the intravenous route was superior to other routes with regard to transgene expression. After intravenous injection of adenovirus expressing human GM-CSF, there was a transient and dose-dependent increase in the serum level of this cytokine. Administration of adenovirus expressing mouse GM-CSF enhanced hematopoiesis in the spleen and bone marrow. CONCLUSION These results indicated that adenoviruses can be used for in vivo cytokine gene therapy but suggested the necessity of taking into consideration the route of administration, the duration of transgene expression, the toxic dose, and host immune reactions.
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Affiliation(s)
- H Kurata
- Department of Molecular and Developmental Biology, the Institute of Medical Science, the University of Tokyo, Japan
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Hogaboam CM, Simpson KJ, Chensue SW, Steinhauser ML, Lukacs NW, Gauldie J, Strieter RM, Kunkel SL. Macrophage inflammatory protein-2 gene therapy attenuates adenovirus- and acetaminophen-mediated hepatic injury. Gene Ther 1999; 6:573-84. [PMID: 10476217 DOI: 10.1038/sj.gt.3300858] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Profound hepatocellular injury is often a consequence of adenovirus-mediated gene therapy or acetaminophen ingestion. The aim of the present study was to examine the role of a CXC chemokine, macrophage inflammatory protein-2 (MIP-2), in the hepatotoxic response by mice infected with adenovirus and challenged with acetaminophen. CD1 mice that received a replication-defective human type 5 adenovirus vector (Ad70-3) intravenously exhibited hepatic injury that peaked at 24 h after infection. In contrast, mice that received a similar adenovirus vector containing a rodent MIP-2 cDNA insert had no hepatic injury at any time after infection. The combination of Ad70-3 infection and an intraperitoneal challenge with 400 mg/kg of acetaminophen was fatal in 50% of the mice, but only 10% of the AdMIP-2 group receiving acetaminophen were similarly affected. Furthermore, AdMIP-2 mice had significantly lower hepatic injury and serum aminotransaminases compared with the Ad70-3 group. However, AdMIP-2 infection in mice lacking the CXC chemokine receptor that binds MIP-2, CXCR2, did not attenuate any of the markers of liver injury after adenovirus and acetaminophen challenge. AdMIP-2 treatment of CD1 mice was also associated with significantly decreased leukocyte infiltration into the liver and an earlier increase in hepatic 3H-thymidine incorporation compared with the control group. Taken together, these data demonstrate that MIP-2 has a protective role in both adenovirus- and acetaminophen-mediated hepatotoxicity, and suggest that MIP-2 may promote rapid hepatic regeneration following acute hepatic injury.
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Affiliation(s)
- C M Hogaboam
- Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602, USA
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Bilbao G, Contreras JL, Gómez-Navarro J, Eckhoff DE, Mikheeva G, Krasnykh V, Hynes T, Thomas FT, Thomas JM, Curiel DT. Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation. Transplantation 1999; 67:775-83. [PMID: 10199723 DOI: 10.1097/00007890-199903270-00001] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.
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Affiliation(s)
- G Bilbao
- Gene Therapy Center, Department of Surgery, University of Alabama at Birmingham, 35294, USA
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46
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Lieber A, He CY, Meuse L, Himeda C, Wilson C, Kay MA. Inhibition of NF-kappaB activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver. J Virol 1998; 72:9267-77. [PMID: 9765474 PMCID: PMC110346 DOI: 10.1128/jvi.72.11.9267-9277.1998] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
NF-kappaB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-kappaB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IkappaBalpha supersuppressor (Ad.IkappaBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-kappaB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IkappaBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 x C3H mice. In the absence of Bcl-2, IkappaBM expression in mouse livers significantly reduced NF-kappaB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IkappaBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-kappaB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-kappaB protection was inactivated by IkappaBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence.
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Affiliation(s)
- A Lieber
- Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98115, USA
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47
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Ye X, Gao GP, Pabin C, Raper SE, Wilson JM. Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse. Hum Gene Ther 1998; 9:2135-42. [PMID: 9759939 DOI: 10.1089/hum.1998.9.14-2135] [Citation(s) in RCA: 50] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The goal of this study is to assess the likelihood that an adenoviral vector disseminated to gonads will be transmitted to offspring. This study is based on the observation that systemically administered vector can be detected in both ovaries and testes, using sensitive nested PCR techniques. Although the extent of vector dissemination to gonads is extremely small, as it is detectable only by nested PCR, it is unclear where it is located within these tissues and whether the DNA is capable of integration and transmission to offspring. A protocol was developed in C3H mice to address this question. Both male and female C3H mice were injected with a high dose of H5.001CBhOTC, an E1- and E4-deleted vector expressing human ornithine transcarbamylase. This dose of vector was sufficient to target 80% of hepatocytes (Gao et al., J. Virol. 1996; 70:8934-8943) and disseminate, at low levels, to both ovaries and testes in 94% of animals as determined by PCR. Vector-administered animals and controls were mated and 814 offspring were evaluated for germ line transmission of the adenoviral vector by DNA hybridization of total cellular DNA extracted from the fetus. Southern blot analysis showed no evidence of germ line transmission in 578 offspring of crosses in which either one or both parents received recombinant adenovirus.
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Affiliation(s)
- X Ye
- Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, University of Pennsylvania School of Medicine, Philadelphia 19104, USA
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Raper SE, Haskal ZJ, Ye X, Pugh C, Furth EE, Gao GP, Wilson JM. Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses. Hum Gene Ther 1998; 9:671-9. [PMID: 9551615 DOI: 10.1089/hum.1998.9.5-671] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Preclinical studies were designed to investigate the feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of nonhuman primates. The vectors used are recombinant adenoviruses deleted in E1 and contain either a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein at nonpermissive temperatures, or a deletion of the E4 region, including open reading frame (ORF) 6. Six 8- to 10-kg baboons underwent femoral artery cannulation, and angiographic techniques were used to introduce vector selectively into either a portion of the right lobe of the liver via a branch of the right hepatic artery or the common hepatic artery. Necropsies were performed at 4, 29, or 61 days. Serial sequential liver biopsies were performed in the baboons that survived 29 or 61 days. In the 2 baboons with vector transduction into the right hepatic artery, X-Gal histochemical analysis of the liver showed evidence of quantitatively increased gene transfer in the targeted lobe; however, gene transfer was present throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was present throughout both livers transduced with the highest dose of vector. No differences were seen in the level of portal inflammation in targeted and untargeted lobes despite the observed qualitative and quantitative differences in gene expression. Southern blot analysis of total cellular DNA isolated from targeted and nontargeted lobes showed similar levels of viral DNA throughout the liver. Polymerase chain reaction (PCR) analysis was able to detect viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. In baboons treated with lower doses of an E1-E4 deleted vector expressing the human ornithine transcarbamylase (OTC) gene, DNA was detectable by nested PCR in liver but not gonads at days 29 and 61. The data suggest that intraarterial administration of recombinant adenoviral E1-E4 deleted vector is feasible and safe. At high doses of vector, widespread dissemination of vector DNA is seen. At low doses, hepatic gene transfer is not associated with vector DNA dissemination to gonads.
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Affiliation(s)
- S E Raper
- Institute for Human Gene Therapy, Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, Philadelphia 19104-6100, USA
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Sato Y, Tanaka K, Lee G, Kanegae Y, Sakai Y, Kaneko S, Nakabayashi H, Tamaoki T, Saito I. Enhanced and specific gene expression via tissue-specific production of Cre recombinase using adenovirus vector. Biochem Biophys Res Commun 1998; 244:455-62. [PMID: 9514856 DOI: 10.1006/bbrc.1997.8087] [Citation(s) in RCA: 76] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
A tissue-specific promoter is potentially valuable for the study of specific gene function and for gene therapy, as it permits a linked cytotoxic or any other gene to be expressed specifically in target cells. The expression levels of such promoters are generally low, and we have therefore developed a novel and general method to enhance the expression level of a tissue-specific promoter while maintaining specificity. We constructed a "regulator" recombinant adenovirus (rAd) producing the site-specific recombinase Cre under the control of the hepatocarcinoma-specific alpha-fetoprotein (AFP) promoter. The rAd was infected to AFP-producing cells together with a "target" rAd containing a Cre-activating potent expression unit. In in vitro experiments, the double infection method gave about 50-fold higher expression than the single rAd infection directly driven by the AFP promoter, while maintaining strict specificity to AFP-producing cells. The enhanced and specific expression was also observed in in vivo tumor models. This method may contribute not only to the establishment of specific gene therapies but also to basic study for elucidating cell-type specific gene functions.
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Affiliation(s)
- Y Sato
- Laboratory of Molecular Genetics, University of Tokyo, Japan
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50
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Hara T, Tan Y, Huang L. In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector. Proc Natl Acad Sci U S A 1997; 94:14547-52. [PMID: 9405650 PMCID: PMC25050 DOI: 10.1073/pnas.94.26.14547] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/1997] [Accepted: 10/27/1997] [Indexed: 02/05/2023] Open
Abstract
Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles ( approximately 100 nm in diameter) with an efficiency approximately 65%. When injected into the portal vein of mice, there were approximately 5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 microg injected DNA. This level of transgene expression was approximately 100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 microg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human alpha1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.
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Affiliation(s)
- T Hara
- Laboratory of Drug Targeting, Department of Pharmacology, School of Medicine, University of Pittsburgh, W1351 Biomedical Science Tower, Pittsburgh, PA 15261, USA
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