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      Gene Delivery by Subconjunctival Injection of Adenovirus in Rats: A Study of Local Distribution, Transgene Duration and Safety

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          Abstract

          Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, β-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short-term ocular transgene expression, without causing hepatic injury and immune activation. Therefore, subconjunctivally administered adenovirus may be a promising gene delivery approach for managing anterior and posterior segment eye diseases requiring short-term therapy.

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          Most cited references36

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          Ocular drug delivery.

          Ocular drug delivery has been a major challenge to pharmacologists and drug delivery scientists due to its unique anatomy and physiology. Static barriers (different layers of cornea, sclera, and retina including blood aqueous and blood-retinal barriers), dynamic barriers (choroidal and conjunctival blood flow, lymphatic clearance, and tear dilution), and efflux pumps in conjunction pose a significant challenge for delivery of a drug alone or in a dosage form, especially to the posterior segment. Identification of influx transporters on various ocular tissues and designing a transporter-targeted delivery of a parent drug has gathered momentum in recent years. Parallelly, colloidal dosage forms such as nanoparticles, nanomicelles, liposomes, and microemulsions have been widely explored to overcome various static and dynamic barriers. Novel drug delivery strategies such as bioadhesive gels and fibrin sealant-based approaches were developed to sustain drug levels at the target site. Designing noninvasive sustained drug delivery systems and exploring the feasibility of topical application to deliver drugs to the posterior segment may drastically improve drug delivery in the years to come. Current developments in the field of ophthalmic drug delivery promise a significant improvement in overcoming the challenges posed by various anterior and posterior segment diseases.
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            Challenges and obstacles of ocular pharmacokinetics and drug delivery.

            Arto Urtti (2006)
            Modern biological research has produced increasing number of promising therapeutic possibilities for medical treatment. These include for example growth factors, monoclonal antibodies, gene knockdown methods, gene therapy, surgical transplantations and tissue engineering. Ocular application of these possibilities involves drug delivery in many forms. Ocular drug delivery is hampered by the barriers protecting the eye. This review presents an overview of the essential factors in ocular pharmacokinetics and selected pharmacological future challenges in ophthalmology.
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              Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

              An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 December 2015
                2015
                : 10
                : 12
                : e0143956
                Affiliations
                [1 ]Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia
                [2 ]Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, Victoria, Australia
                [3 ]Department of Ophthalmology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
                [4 ]Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
                [5 ]School of Medicine, National Yang-Ming University, Taipei, Taiwan
                [6 ]National Defense Medical Center, Taipei, Taiwan
                [7 ]Yuh-Ing Junior College of Health Care & Management, Kaohsiung, Taiwan
                University of Florida, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GSL JHW MHT YSB. Performed the experiments: GSL JHW PJT HET YSB. Analyzed the data: GSL JHW JHL YSB. Contributed reagents/materials/analysis tools: SJS HCL MHT GJD. Wrote the paper: GSL JHW JHL YSB.

                Article
                PONE-D-15-38511
                10.1371/journal.pone.0143956
                4671571
                26642208
                1e4cc0ff-00f5-47e7-a6e7-5005feac60a8
                © 2015 Liu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 1 September 2015
                : 11 November 2015
                Page count
                Figures: 5, Tables: 2, Pages: 15
                Funding
                This work was supported by grants from National Science Council, Taiwan (NSC100-2314-B-075B-006), Kaohsiung Veterans General Hospital (VGHKS 96-075, 98-062, 99-062, 101-056) and the National Health and Medical Research Council of Australia (NHMRC #1061912). The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government.
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                Research Article
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                All relevant data are within the paper and its Supporting Information files.

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