title: Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.
abstract: Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 degrees C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells.
authors.affiliation: Regulon Inc., Biopole Epalinges, Les Croisettes 22, CH-1066 Epalinges, Switzerland. Kenneth.Lundstrom@mepnet.org
authors.forename: Kenneth
authors.initials: K
authors.lastname: Lundstrom
authors.forename: Alessandra
authors.initials: A
authors.lastname: Abenavoli
authors.forename: Antonio
authors.initials: A
authors.lastname: Malgaroli
authors.forename: Markus U
authors.initials: MU
authors.lastname: Ehrengruber
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000473:pathology / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism / Q000821:virology; D017354:Point Mutation; D051381:Rats; D012672:Semliki forest virus / Q000235:genetics*; D013696:Temperature; D013997:Time Factors; D019076:Transgenes
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins