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Gene Transfer into Hepatocytes Mediated by Helper Virus-Free HSV/AAV Hybrid Vectors

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Abstract

Background

Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo.

Materials and Methods

HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase−/−) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity.

Results

Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (∼2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2−/− mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier.

Conclusions

HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.

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Acknowledgments

We thank Drs. K. Ponder, K. Zaret, H. Federoff, and F. Boyce for plasmid constructs, Dr. B. Knowles for the NPLC cells, Dr. F. Smith for collaborative insight, and J. Elfar for critically reading the manuscript. This work was supported by the Swiss National Science Foundation and the American Liver Foundation (C.F.), NINDS NS24279, NCI CA69246, NIDCD DC002281 (X.O.B.).

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Author notes

  1. Cornel Fraefel

    Present address: Institute of Virology, University of Zurich, Winterthurerstrasse 266a, CH-8057, Zurich, Switzerland

Authors and Affiliations

  1. Molecular Neurogenetics Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

    Cornel Fraefel, David R. Jacoby & Xandra O. Breakefield

  2. Division of Endocrinology, The Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Cornel Fraefel, Christopher Lage, Harold Hilderbrand & Joseph A. Majzoub

  3. Howard Hughes Medical Institute, The Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Fred W. Alt

  4. Heritable Disorders Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA

    Janice Y. Chou

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Correspondence to Cornel Fraefel.

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Fraefel, C., Jacoby, D.R., Lage, C. et al. Gene Transfer into Hepatocytes Mediated by Helper Virus-Free HSV/AAV Hybrid Vectors. Mol Med 3, 813–825 (1997). https://doi.org/10.1007/BF03401718

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