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.

Differential Roles of NR2A- and NR2B-Containing NMDA Receptors in Activity-Dependent Brain-Derived Neurotrophic Factor Gene Regulation and Limbic Epileptogenesis

“…To acutely interfere NR2A-and NR2B-dependent synaptic signaling, we used a Semliki Forest virus (SFV) to overexpress the carboxyl cytoplasmic tail (838 -1464 aa) of NR2A or of NR2B (839 -1482 aa) fused with EGFP in cultured hippocampal neurons. SFV confers several advantages over other gene delivery approaches, including easy and fast generation of recombinant viral particles, rapid and high-level transgene expression, and efficiently and preferentially infecting neurons rather than non-neuronal cells (Ehrengruber, 2002;Lundstrom et al, 2003). Electrophysiological recording showed that overexpression of NR2A tail , NR2B tail , or EGFP in cultured neurons did not affect of the NMDA-induced peak current, suggesting that these expressed peptides do not perturb NMDAR channel opening (supplemental Fig.…”

Astrocytes Display Complex and Localized Calcium Responses to Single-Neuron Stimulation in the Hippocampus

“…Farnesylated mCherry (mCherry-f) was created by cloning the farnesylation sequence of Ha-Ras onto the C terminus of mCherry (from R. Tsien, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA). Genes for ChR2-Venus and mCherry-f were each cloned 3Ј to a viral subgenomic promoter in a modified SFV vector (SFV-PD) (Lundstrom et al, 2003;Zhou et al, 2007). The SFV-PD vector containing eGFP-f alone was described previously (Haber et al, 2006).…”

Biology and application of alphaviruses in gene therapy

“…Similar less cytotoxic and temperaturesensitive vectors have also been developed for SFV, where typically a triple mutant vector substantially prolongs the transgene expression time in cell cultures up to at least 20 days. 32 Recently, a novel SFV expression vector based on the avirulent A7 strain was developed, which showed a temperature-dependent expression pattern in hippocampal slice cultures. 33 At 371C, the GFP expression pattern was mostly glial specific, whereas at 311C, the majority of GFP-positive cells were of neuronal origin.…”