title: Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS.
abstract: The development and implementation of direct gene transfer technologies for the study and treatment of chronic CNS disorders inherently requires consideration of vector safety. Virus-based vectors represent the most efficient modalities but harbor the potential to induce vigorous innate and adaptive immune responses when administered in vivo. These responses can arise because of virus particle components, resultant viral gene expression, and/or transgene expression. In the current study, we describe the innate responses elicited upon stereotactic delivery of herpes simplex virus type 1-based amplicon vectors. C57BL/6 mice were injected with sterile saline, beta-galactosidase-expressing amplicon (HSVlac) packaged by a conventional helper virus-based technique, or helper virus-free HSVlac. After killing the mice at either 1 or 5 days after transduction, we analyzed them by immunocytochemistry and quantitative RT-PCR for various chemokine, cytokine, and adhesion molecule gene transcripts. All injections induced inflammation, with blood/brain barrier opening on day 1 that was enhanced with both amplicon preparations as compared with saline controls. By day 5, mRNA levels for the pro-inflammatory cytokines (IL-1beta, TNF-alpha, IFN-gamma), chemokines (MCP-1, IP-10), and an adhesion molecule (ICAM-1) had returned to baseline in saline-injected mice and to near-baseline levels in helper virus-free amplicon groups. In contrast, mice injected with helper virus-packaged amplicon stocks elicited elevated inflammatory molecule expression and immune cell infiltration even at day 5. In aggregate, we demonstrate that helper virus-free amplicon preparations exhibit a safer innate immune response profile, presumably as a result of the absence of helper virus gene expression, and provide support for future amplicon-based CNS gene transfer strategies.
authors.affiliation: Departments of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. john_olschowka@urmc.rochester.edu
authors.forename: John A
authors.initials: JA
authors.lastname: Olschowka
authors.forename: William J
authors.initials: WJ
authors.lastname: Bowers
authors.forename: Sean D
authors.initials: SD
authors.lastname: Hurley
authors.forename: Michael A
authors.initials: MA
authors.lastname: Mastrangelo
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Federoff
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002490:Central Nervous System / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000096:biosynthesis; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000096:biosynthesis; D018076:DNA, Complementary / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D007150:Immunohistochemistry; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D007371:Interferon-gamma / Q000096:biosynthesis; D007375:Interleukin-1 / Q000096:biosynthesis; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000473:pathology; D009504:Neutrophils / Q000166:cytology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D018932:Chemokine CCL2; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D018076:DNA, Complementary; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma