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Mao J, Xi X, Kapranov P, Dong B, Firrman J, Xu R, Xiao W. In vitro and In vivo Model Systems for Hemophilia A Gene Therapy. ACTA ACUST UNITED AC 2013; Suppl 1. [PMID: 25401041 PMCID: PMC4229687 DOI: 10.4172/2157-7412.s1-014] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Hemophilia A is a hereditary disorder caused by various mutations in factor VIII gene resulting in either a severe deficit or total lack of the corresponding activity. Recent success in gene therapy of a related disease, hemophilia B, gives new hope that similar success can be achieved for hemophilia A as well. To develop a gene therapy strategy for the latter, a variety of model systems are needed to evaluate molecular engineering of the factor VIII gene, vector delivery efficacy and safety-related issues. Typically, a tissue culture cell line is the most convenient way to get a preliminary glimpse of the potential of a vector delivery strategy. It is then followed by extensive testing in hemophilia A mouse and dog models. Newly developed hemophilia A sheep may provide yet another tool for evaluation of factor VIII gene delivery vectors. Hemophilia models based on other species may also be developed since hemophiliac animals have been identified or generated in rat, pig, cattle and horse. Although a genetic nonhuman primate hemophilia A model has yet to be developed, the non-genetic hemophilia A model can also be used for special purposes when specific questions need to be addressed that cannot not be answered in other model systems. Hemophilia A is caused by a functional deficiency in the factor VIII gene. This X-linked, recessive bleeding disorder affects approximately 1 in 5000 males [1–3]. Clinically, it is characterized by frequent and spontaneous joint hemorrhages, easy bruising and prolonged bleeding time. The coagulation activity of FVIII dictates severity of the clinical symptoms. Approximately 50% of all cases are classified as severe with less than 1% of normal levels of factor VIII detected [4]. This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding. In contrast, patients with 5–30% of normal factor VIII activity exhibit mild clinical manifestations.
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Affiliation(s)
- Jianhua Mao
- Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China ; Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA
| | - Xiaodong Xi
- Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | | | - Biao Dong
- Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA
| | - Jenni Firrman
- Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA
| | - Ruian Xu
- Institute of Molecular Medicine, Molecular Medicine Engineering Research Center, Huaqiao University, Quanzhou 362021, China
| | - Weidong Xiao
- Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA
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2
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Matsui H. Endothelial progenitor cell-based therapy for hemophilia A. Int J Hematol 2012; 95:119-24. [PMID: 22314304 DOI: 10.1007/s12185-012-1015-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2011] [Revised: 01/19/2012] [Accepted: 01/20/2012] [Indexed: 01/02/2023]
Abstract
As shown by the results of both pre-clinical and clinical studies reported in past decades, the goal of establishing an effective and successful gene therapy for hemophilia A remains feasible and realistic. However, at this time, no single approach has been shown to be clearly superior, and a number of recurring challenges remain to be overcome. Given the persistent problems presented by the host immune response to systemic in vivo gene delivery, and the additional obstacles of inadequate transgene delivery and expression, we propose a re-evaluation of an ex vivo gene transfer approach that utilizes a genetically modified stem cell population. In this strategy, autologous blood outgrowth endothelial progenitor cells are obtained from hemophilic animals, into which a normal copy of the factor VIII gene is introduced via an engineered virus. Cell numbers are expanded in culture prior to their re-implantation under the skin of the hemophilic animals in an artificially developed supporting environment. Follow-up assessment of the treatment involves the general evaluation of clotting activity, the specific measurement of factor VIII levels in the blood, and clinical observation.
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Affiliation(s)
- Hideto Matsui
- Department of Regulatory Medicine for Thrombosis, Nara Medical University, 840 Shijo-cho, Kashihara Nara, 634-8521, Japan.
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Jinturkar KA, Rathi MN, Misra A. Gene Delivery Using Physical Methods. CHALLENGES IN DELIVERY OF THERAPEUTIC GENOMICS AND PROTEOMICS 2011:83-126. [DOI: 10.1016/b978-0-12-384964-9.00003-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Lillicrap D. Hemophilia Gene Therapy: An Overview. TEXTBOOK OF HEMOPHILIA 2010:226-230. [DOI: 10.1002/9781444318555.ch35] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Kamimura K, Zhang G, Liu D. Image-guided, intravascular hydrodynamic gene delivery to skeletal muscle in pigs. Mol Ther 2010; 18:93-100. [PMID: 19738603 PMCID: PMC2805042 DOI: 10.1038/mt.2009.206] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2009] [Accepted: 08/06/2009] [Indexed: 12/12/2022] Open
Abstract
Development of an effective, safe, and convenient method for gene delivery to muscle is a critical step toward gene therapy for muscle-associated diseases. Toward this end, we have explored the possibility of combining the image-guided catheter insertion technique with the principle of hydrodynamic delivery to achieve muscle-specific gene transfer in pigs. We demonstrate that gene transfer efficiency of the procedure is directly related to flow rate, injection pressure, and injection volume. The optimal gene delivery was achieved at a flow rate of 15 ml/second with injection pressure of 300 psi and injection volume equal to 1.5% of body weight. Under such a condition, hydrodynamic injection of saline containing pCMV-Luc (100 microg/ml) resulted in luciferase activity of 10(6) to 10(7) relative light units (RLU)/mg of proteins extracted from the targeted muscle 5 days after hydrodynamic gene delivery. Result from immunohistochemical analysis revealed 70-90% transfection efficiency of muscle groups in the hindlimb and persistent reporter gene expression for 2 months in transfected cells. With an exception of transient edema and elevation of creatine phosphokinase, no permanent tissue damage was observed. These results suggest that the image-guided, intravenous hydrodynamic delivery is an effective and safe method for gene delivery to skeletal muscle.
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Affiliation(s)
- Kenya Kamimura
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
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A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells. Blood 2009; 114:677-85. [PMID: 19458355 PMCID: PMC9710235 DOI: 10.1182/blood-2009-03-202267] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Under certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice. We used lentiviral vectors to deliver the canine FVIII transgene to neonatal hemophilic mice and demonstrated that induction of long-term FVIII tolerance could be achieved. Hemophilia A mice are capable of mounting a robust immune response to FVIII after neonatal gene transfer, and tolerance induction is dependent on the route of delivery and type of promoter used. High-level expression of FVIII was not required for tolerance induction and, indeed, tolerance developed in some animals without evidence of detectable plasma FVIII. Tolerance to FVIII could be adoptively transferred to naive hemophilia recipient mice, and FVIII-stimulated splenocytes isolated from tolerized mice expressed increased levels of interleukin-10 and decreased levels of interleukin-6 and interferon-gamma. Finally, induction of FVIII tolerance mediated by this protocol is associated with a FVIII-expandable population of CD4(+)CD25(+)Foxp3(+) regulatory T cells.
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Kren BT, Yin W, Key NS, Hebbel RP, Steer CJ. Blood Outgrowth Endothelial Cells as a Vehicle for Transgene Expression of Hepatocyte-Secreted Proteins viaSleeping Beauty. ACTA ACUST UNITED AC 2009; 14:97-104. [PMID: 17497366 DOI: 10.1080/10623320701346932] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The therapeutic use of autologous cells with the capacity for extensive in vitro expansion and manipulation prior to host administration has been an area of significant investigation over the last decade. Blood outgrowth endothelial cells (BOECs) are derived from the circulation and exhibit proliferative growth, in vivo engraftment, and survival characteristics for long-term expression of endogenously secreted proteins, such as factor VIII (FVIII). The authors describe a modified method for the isolation, culture, and expansion of these cells that is readily accomplished using standard laboratory methods. Using a commercially available transfection reagent, approximately 30% of these primary cells can be routinely transfected with the nonviral Sleeping Beauty transposon for long-term, stable transgene expression. Moreover, the results indicate that these cells have the ability to secrete functionally active proteins that are synthesized endogenously by hepatocytes and require post-translational modification including alpha1-antitrypsin and clotting factors VII and IX. This, coupled with their notably long half-life of years, suggests that these cells may provide an appropriate vehicle for secretion of a variety of proteins produced by different cell types in vivo. Thus, BOECs have the potential to provide clinically relevant secreted proteins for diseases other than those of endothelial origin.
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Affiliation(s)
- Betsy T Kren
- Department of Medicine, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
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Kren BT, Unger GM, Sjeklocha L, Trossen AA, Korman V, Diethelm-Okita BM, Reding MT, Steer CJ. Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice. J Clin Invest 2009; 119:2086-99. [PMID: 19509468 DOI: 10.1172/jci34332] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2007] [Accepted: 04/22/2009] [Indexed: 12/16/2022] Open
Abstract
Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.
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Affiliation(s)
- Betsy T Kren
- Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA
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Wooddell CI, Reppen T, Wolff JA, Herweijer H. Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery. J Gene Med 2008; 10:551-63. [PMID: 18330848 DOI: 10.1002/jgm.1179] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND To properly study gene expression in vivo, often long-term expression is desired. Previous studies using plasmid DNA (pDNA) vectors have typically resulted in short-term expression. Here, we evaluated combinations of the albumin promoter with different enhancers and untranslated regions for liver-specific expression in mice. METHODS A series of pDNA secreted alkaline phosphatase (SEAP) reporter gene expression vectors was constructed using the albumin promoter and various other expression cassette elements. Each was evaluated for level and duration of SEAP expression in mice following hydrodynamic tail vein delivery. RESULTS Sustained liver expression was obtained from vectors combining the albumin promoter with an albumin 3' untranslated region (3'UTR). The level of expression was increased by inclusion of enhancers and a 5' intron. The optimal expression vector consisted of the albumin promoter combined with an alpha-fetoprotein MERII enhancer, 5' intron from the factor IX gene, and the 3'UTR from the albumin gene including intron 14. With this vector, SEAP reporter gene expression levels remained high for 1 year, at levels comparable to those obtained from the cytomegalovirus (CMV) promoter on day 1. Expression of human apolipoprotein E3 (hApoE) in ApoE knockout mice provided a dose-dependent correction of their hypercholesterolemia. CONCLUSIONS Liver-specific sustained transgene expression can be obtained at very high levels from optimized pDNA vectors, without the use of integration systems. Such vectors will further facilitate biological studies of genes in vivo and may find application in gene therapy.
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Wu Z, Sun J, Zhang T, Yin C, Yin F, Van Dyke T, Samulski RJ, Monahan PE. Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose. Mol Ther 2007; 16:280-9. [PMID: 18059373 DOI: 10.1038/sj.mt.6300355] [Citation(s) in RCA: 128] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.
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Affiliation(s)
- Zhijian Wu
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352, USA
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11
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Abstract
Efficient and safe methods for delivering genetic materials into cells must be developed before the clinical potential of gene therapy can be fully realized. Recently, hydrodynamic gene delivery using a rapid injection of a relatively large volume of DNA solution has opened up a new avenue for gene therapy studies in vivo. This method is superior to the existing delivery systems because of its simplicity, efficiency, and versatility. Wide success in applying hydrodynamic principles to delivery of DNA, RNA, proteins, and synthetic compounds, into the cells in various tissues of small animals, has inspired the recent attempts at establishing a hydrodynamic procedure for clinical use. In this review, we provide an overview of the theory and practice of hydrodynamic gene delivery so as to aid researchers for the use of this method in their pre-clinical and translational gene therapy studies.
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Affiliation(s)
- Takeshi Suda
- 1Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15261, USA
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12
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Picanço V, Heinz S, Bott D, Behrmann M, Covas DT, Seifried E, Tonn T. Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter. Cytotherapy 2007; 9:785-94. [PMID: 17917890 DOI: 10.1080/14653240701656053] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
BACKGROUND Lentiviral vectors have the capacity to transduce stably non-dividing, differentiated and undifferentiated cells of various tissues, including liver. To obtain high-level expression of transgenes, vectors often rely on viral promoters. However, recent data suggest that the supraphysiologic expression from ubiquitous viral promoters may not be beneficial and harbor the risk of oncogene activation. Therefore this study explored the lentiviral-mediated expression of human coagulation factor VIII (FVIII) driven by the physiologic FVIII gene promoter (FVIII-p), the liver-specific human alpha-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1alpha promoter (EF1alpha-p) and the viral CMV promoter. METHODS Hepatic and non-hepatic cell lines were stably transduced with lentiviral vectors encoding FVIIIdelB and EGFP. To compare the different promoters, lentiviral vectors were cloned to drive FVIII expression from FVIII-p, EF1alpha-p, hAAT-p and CMV-p. RESULTS As expected, the strong viral CMV-p and the ubiquitous EF1alpha-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/10(6) cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-alpha promoter 0.30 IU/mL/10(6) cells for 293T, 0.04 for HepG2, 2.75 for SK-Hep, 0.46 for Hepa1-6). While the hAAT-p resulted in low FVIII levels (0.10 IU/mL/10(6)cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/10(6)cells in Hepa1-6 and 0.44 in SK-Hep). DISCUSSION These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.
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Affiliation(s)
- V Picanço
- Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Johann Wolfgang Goethe University Clinics, Frankfurt/Main, Germany
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Matsui H, Shibata M, Brown B, Labelle A, Hegadorn C, Andrews C, Hebbel RP, Galipeau J, Hough C, Lillicrap D. Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors. Stem Cells 2007; 25:2660-9. [PMID: 17615271 DOI: 10.1634/stemcells.2006-0699] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- Hideto Matsui
- Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada
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Yeikilis R, Gal S, Kopeiko N, Paizi M, Pines M, Braet F, Spira G. Hydrodynamics based transfection in normal and fibrotic rats. World J Gastroenterol 2006; 12:6149-55. [PMID: 17036386 PMCID: PMC4088108 DOI: 10.3748/wjg.v12.i38.6149] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated.
METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection.
RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect.
CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced.
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Affiliation(s)
- Rita Yeikilis
- Department of Anatomy and Cell Biology, Faculty of Medicine, POB 9649, Haifa 31096, Israel
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15
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Affiliation(s)
- C Hough
- Department of Pathology and Molecular Medicine, Richardson Laboratories, Queen's University, Kingston, Ontario, Canada
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16
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Rawle FEM, Shi CX, Brown B, McKinven A, Tinlin S, Graham FL, Hough C, Lillicrap D. Heterogeneity of the immune response to adenovirus-mediated factor VIII gene therapy in different inbred hemophilic mouse strains. J Gene Med 2005; 6:1358-68. [PMID: 15493040 DOI: 10.1002/jgm.624] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND The development of anti-factor VIII (FVIII) antibodies (inhibitors) is a critical concern when considering gene therapy as a potential treatment modality for hemophilia A. We used a hemophilia A mouse model bred on different genetic backgrounds to explore genetically controlled differences in the immune response to FVIII gene therapy. METHODS C57BL/6 FVIII knockout (C57-FVIIIKO) mice were bred with normal BALB/c (BAL) mice, to generate a recombinant congenic BAL-FVIIIKO model of hemophilia A. Early generation adenoviral (Ad) vectors containing the canine FVIII B-domain-deleted transgene under the control of either the CMV promoter or a tissue-restricted (TR) promoter were administered to C57-FVIIIKO, C57xBAL(F1)-FVIIIKO crosses, and BAL-FVIIIKO mice. FVIII expression, inhibitor development, inflammation, and vector-mediated toxicity were assessed. RESULTS In response to administration of Ad-CMV-cFVIII, C57-FVIIIKO mice attain 3-fold higher levels of FVIII expression than BAL-FVIIIKO. All strains injected with Ad-CMV-FVIII displayed FVIII expression lasting only 2 weeks, with associated inhibitor development. C57-FVIII-KO mice that received Ad-TR-FVIII expressed FVIII for 12 months post-injection, whereas FVIII expression was limited to 1 week in C57xBAL(F1)-FVIIIKO and BAL-FVIIIKO mice. This loss of expression was associated with anti-FVIII inhibitor development. BAL-FVIIIKO mice showed increased hepatotoxicity with alanine aminotransferase levels reaching 4-fold higher levels than C57-FVIIIKO mice. However, C57-FVIIIKO mice initiate a more rapid and effective cell-mediated clearance of virally transduced cells than BAL-FVIIIKO, as evidenced by real-time PCR analysis of transduced tissues. Overall, strain-dependent differences in the immune response to FVIII gene delivery were only noted in the adaptive response, and not in the innate response. CONCLUSIONS Our results indicate that the genetic background of the murine model of hemophilia A influences FVIII expression levels, the development of anti-FVIII inhibitors, clearance of transduced cells, and the severity of vector-mediated hepatotoxicity.
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Affiliation(s)
- Fiona E M Rawle
- Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6
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Kobayashi N, Nishikawa M, Takakura Y. The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels. Adv Drug Deliv Rev 2005; 57:713-31. [PMID: 15757757 DOI: 10.1016/j.addr.2004.12.006] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2004] [Accepted: 12/18/2004] [Indexed: 10/25/2022]
Abstract
Hydrodynamics-based gene delivery, involving a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), gives a significantly high level of transgene expression in vivo. This has attracted a lot of attention and has been used very frequently as an efficient, simple and convenient transfection method for laboratory animals. Until recently, however, little information has been published on the pharmacokinetics of the injected DNA molecules and of the detailed mechanisms underlying the efficient gene transfer. We and other groups have very recently demonstrated that the mechanism for the hydrodynamics-based gene transfer would involve, in part, the direct cytosolic delivery of pDNA through the cell membrane due to transiently enhanced permeability. Along with the findings in our series of studies, this article reviews the cumulative reports and other intriguing information on the controlled pharmacokinetics of naked pDNA in the hydrodynamics-based gene delivery. In addition, we describe various applications reported so far, as well as the current attempts and proposals to develop novel gene medicines for future gene therapy using the concept of the hydrodynamics-based procedure. Furthermore, the issues associated with the clinical feasibility of its seemingly invasive nature, which is probably the most common concern about this hydrodynamics-based procedure, are discussed along with its future prospects and challenges.
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Affiliation(s)
- Naoki Kobayashi
- Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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Ye P, Thompson AR, Sarkar R, Shen Z, Lillicrap DP, Kaufman RJ, Ochs HD, Rawlings DJ, Miao CH. Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice. Mol Ther 2005; 10:117-26. [PMID: 15233948 DOI: 10.1016/j.ymthe.2004.04.009] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2004] [Accepted: 04/14/2004] [Indexed: 11/17/2022] Open
Abstract
The development of antibodies to a previously unexpressed protein product may limit the success of human gene therapy approaches. We inserted B-domain-deleted factor VIII (FVIII) cDNA of human, canine, or murine origin into the multiple cloning site of a liver-specific vector, pBS-HCRHPI-A, to yield plasmids pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA, respectively. Fifty micrograms of each plasmid in 2 ml of solution was rapidly injected into the tail vein of three groups of hemophilia A mice. Factor VIII levels ranging from 3 to 12 IU/ml were obtained from all three groups (normal is 1 IU/ml in human plasma) 3 days after treatment. These initial very high levels of functional human, canine, or murine factor VIII, however, fell gradually to undetectable levels within 2-3 weeks, and their disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Notably, this immune response occurred independent of the species of origin of the exogenous factor VIII. Antibody titers to factor VIII were detected beginning at 2 weeks, reached a plateau and remained at high levels for over 6 months. The majority of anti-hFVIII IgG was IgG1 isotype specific, suggesting a humoral response mediated by Th2-induced signals. Consistent with this idea, in a separate group of mice treated with pBS-HCRHPI-FVIIIA, transient immunosuppression by cyclophosphamide significantly delayed (5/6) or abolished (1/6) inhibitory antibody formation against the transgene.
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Affiliation(s)
- Peiqing Ye
- Department of Pediatrics and Department of Medicine, University of Washington, Seattle, WA 98195, USA
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Miao CH. A novel gene expression system: non-viral gene transfer for hemophilia as model systems. ADVANCES IN GENETICS 2005; 54:143-77. [PMID: 16096011 DOI: 10.1016/s0065-2660(05)54007-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
It is highly desirable to generate tissue-specific and persistently high-level transgene expression per genomic copy from gene therapy vectors. Such vectors can reduce the cost and preparation of the vectors and reduce possible host immune responses to the vector and potential toxicity. Many gene therapy vectors have failed to produce therapeutic levels of transgene because of inefficient promoters, loss of vector or gene expression from episomal vectors, or a silencing effect of integration sites on integrating vectors. Using in vivo screening of vectors incorporating many different combinations of gene regulatory sequences, liver-specific, high-expressing vectors to accommodate factor IX, factor VIII, and other genes for effective gene transfer have been established. Persistent and high levels of factor IX and factor VIII gene expression for treating hemophilia B and A, respectively, were achieved in mouse livers using hydrodynamics-based gene transfer of naked plasmid DNA incorporating these novel gene expression systems. Some other systems to prolong or stabilize the gene expression following gene transfer are also discussed.
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Affiliation(s)
- Carol H Miao
- Department of Pediatrics, University of Washington and Children's Hospital and Regional Medical Center, Seattle, Washington 98195, USA
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Al-Dosari MS, Knapp JE, Liu D. Hydrodynamic Delivery. NON-VIRAL VECTORS FOR GENE THERAPY, SECOND EDITION: PART 2 2005; 54:65-82. [PMID: 16096008 DOI: 10.1016/s0065-2660(05)54004-5] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Hydrodynamic delivery has emerged as a near-perfect method for intracellular DNA delivery in vivo. For gene delivery to parenchymal cells, only essential DNA sequences need to be injected via a selected blood vessel, eliminating safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is capable of reaching cells in the different tissues accessible to the blood. Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution into the incompressible blood in the circulation to overcome the physical barriers of endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to the delivery of DNA, this method is useful for the efficient intracellular delivery of RNA, proteins, and other small compounds in vivo. This review discusses the development, current application, and clinical potential of hydrodynamic delivery.
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Affiliation(s)
- Mohammed S Al-Dosari
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15261, USA
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Miao CH, Ye X, Thompson AR. High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors. Hum Gene Ther 2004; 14:1297-305. [PMID: 14503965 DOI: 10.1089/104303403322319381] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Two liver-specific nonviral gene transfer vectors have been developed to accommodate heterologous genes. The expression cassettes contain (1) a hepatic locus control region from the apolipoprotein E (ApoE) gene (HCR), (2) a liver-specific alpha(1)-antitrypsin promoter (HP), (3) a 1.4-kb truncated factor IX first intron (I) or a synthetic minx intron (mI), (4) a multiple cloning site (MCS) for inserting cDNA sequences, and (5) a bovine growth hormone polyadenylation signal (bpA) to make pBS-HCRHPI-A or pBS-HCRHPmI-A. These vectors were first evaluated with reporter genes encoding human factor IX (hFIX) and green fluorescent protein (GFP). hFIX constructs, pBS-HCRHPI-FIXA and control pBS-HCRHP-FIXIA with the hFIX intron in its native position, produced comparable hFIX gene expression levels (0.5-5 microg/ml) 6 months after naked DNA transfer to mice, whereas the factor IX level from pBS-HCRHPmI-FIXA averaged about 50% lower. RT-PCR analysis of the mRNA indicated that introns inserted upstream from the cDNA were correctly processed and spliced. GFP expression was detected in 15-30% of the hepatocytes in pBS-HCRHPI-GFPA-treated mice. Next, a B domain-deleted human factor VIII (hFVIII) cDNA was inserted into the modified vectors. High-level hFVIII expression (up to 750 ng/ml) was achieved initially in both C57BL/6 mice and Rag2 mice. Moreover, therapeutic levels of hFVIII (20-310 ng/ml) circulated in Rag2 mice 6 months after treatment. These liver-specific gene expression cassettes can deliver a large, heterologous gene such as hFVIII cDNA to achieve high-level, persistent transgene expression after in vivo hepatic gene therapy.
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Affiliation(s)
- Carol H Miao
- Department of Pediatrics and Medicine, University of Washington, WA 98195, USA.
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Brown BD, Shi CX, Powell S, Hurlbut D, Graham FL, Lillicrap D. Helper-dependent adenoviral vectors mediate therapeutic factor VIII expression for several months with minimal accompanying toxicity in a canine model of severe hemophilia A. Blood 2004; 103:804-10. [PMID: 14512318 DOI: 10.1182/blood-2003-05-1426] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractTwo helper-dependent (HD) adenoviral vectors encoding a canine factor VIII B-domain–deleted transgene (cFVIII) were constructed and evaluated in 4 hemophilia A dogs. One vector was regulated by the cytomegalovirus (CMV) promoter (HD-CMV-cFVIII), while the other vector contained a tissue-restricted promoter comprised of the human FVIII proximal promoter with an upstream concatemer of 5 hepatocyte nuclear factor 1 binding sites (HD-HNF-cFVIII). We detected no toxicity at low dose (5 × 1011 vp/kg), but at higher vector doses (> 1 × 1012 vp/kg) transient hepatotoxicity and thrombocytopenia were observed. Low-level increases in FVIII activity were detected in all 3 HD-HNF-cFVIII–treated dogs, which corresponded with decreased whole blood clotting times. None of the animals receiving the HD-HNF-cFVIII vector developed FVIII inhibitors, and in 1 of the 3 animals, FVIII activity was sustained for over 6 months after treatment. One animal, which received the HD-CMV-cFVIII vector, achieved peak levels of FVIII above 19 000 mU/mL, but FVIII activity disappeared within 1 week, coincident with the development of a potent anti–canine FVIII antibody response. This study supports previous demonstrations of improved safety using HD gene transfer and suggests that these vectors can provide transient FVIII expression with minimal, acute toxicity in the absence of inhibitor formation.
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Affiliation(s)
- Brian D Brown
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, ON, Canada K7L 3N6
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Brown BD, Shi CX, Rawle FEM, Tinlin S, McKinven A, Hough C, Graham FL, Lillicrap D. Factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia A mice. J Thromb Haemost 2004; 2:111-8. [PMID: 14717974 DOI: 10.1111/j.1538-7836.2004.00552.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. OBJECTIVE To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. METHODS HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation. RESULTS When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. CONCLUSIONS HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.
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Affiliation(s)
- B D Brown
- Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada
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Abstract
Hydrodynamic delivery is an efficient and inexpensive procedure to deliver a wide range of nucleic acids to hepatic tissues and other organs in vivo. The successful application of hydrodynamic delivery is dependent on the rapid injection of a large aqueous volume containing DNA, RNA or other molecules into the vasculature of the liver. In this review, the development of the procedures for hydrodynamic delivery will be described and the parameters necessary for attaining maximal gene expression will be highlighted. A review of the mechanisms for transfecting hepatocytes, as well as potential uses of this approach in various research and clinical applications, will also be discussed.
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Affiliation(s)
- Bradley L Hodges
- Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701, USA.
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Youil R, Toner TJ, Su Q, Casimiro D, Shiver JW, Chen L, Bett AJ, Rogers BM, Burden EC, Tang A, Chen M, Emini EA, Kaslow DC, Aunins JG, Altaras NE. Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses. Hum Gene Ther 2003; 14:1017-34. [PMID: 12869219 DOI: 10.1089/104303403766682278] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.
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Affiliation(s)
- Rima Youil
- Merck & Company, West Point, PA 19486, USA.
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