Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2B or 2A subunits

Abstract

Because of the numerous types of neurons in the brain, and particularly the forebrain, neuron type-specific expression will benefit many potential applications of direct gene transfer. The two most promising approaches for achieving neuron type-specific expression are targeted gene transfer to a specific type of neuron and using a neuron type-specific promoter. We previously developed antibody-mediated targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors by modifying glycoprotein C (gC) to replace the heparin binding domain, which mediates the initial binding of HSV-1 particles to many cell types, with the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. We showed that a chimeric gC--ZZ protein is incorporated into vector particles and binds IgG. As a proof-of-principle for antibody-mediated targeted gene transfer, we isolated complexes of these vector particles and an anti-NMDA NR1 subunit antibody, and demonstrated targeted gene transfer to neocortical cells that contain NR1 subunits. However, because most forebrain neurons contain NR1, we obtained only a modest increase in the specificity of gene transfer, and this targeting specificity is of limited utility for physiological experiments. Here, we report efficient antibody-mediated targeted gene transfer to NMDA NR2B- or NR2A-containing cells in rat postrhinal cortex, and a neuron-specific promoter further restricted recombinant expression to neurons. Of note, because NR2A-containing neurons are relatively rare, these results show that antibody-mediated targeted gene transfer with HSV-1 vectors containing neuron type-specific promoters can restrict recombinant expression to specific types of forebrain neurons of physiological significance.

Targeted gene transfer of different genes to presynaptic and postsynaptic neocortical neurons connected by a glutamatergic synapse

“…We previously quantified the titer of vector genomes (VG/ml), and the packaging efficiency (VG/ml / IVP/ml), for pVGLUT1lac, pINS-TH-NFHlac, and specific vector stocks for antibody-mediated targeted gene transfer, using a PCR assay; the VG/ml titer, and the packaging efficiencies, for these stocks were similar to a number of other vectors we have studied (Cao et al, 2010; Cao et al, 2011; Gao et al, 2007; Rasmussen et al, 2007; Yang et al, 2001; Zhang et al, 2000). We did not repeat the VG/ml assay here because the vectors used here are similar to previously studied vectors.…”

“…In this study, the presynaptic vector was delivered by standard, untargeted gene transfer, and this vector used the VGLUT1 promoter to restrict expression to VGLUT1-containing glutamatergic neurons (Rasmussen et al, 2007; Zhang and Geller, 2010). The presynaptic vector could be delivered into specific neuron types using targeted gene transfer; we established antibody-mediated targeted gene transfer to NR1-, NR2A-, or NR2B-containing neurons (Cao et al, 2010; Cao et al, 2011), and ligand-mediated targeted gene transfer to neurons containing specific neurotrophic factor receptors (Cao et al, 2008; Wang et al, 2005). Further, expression from the presynaptic vector could be restricted to specific glutamatergic neuron subtypes by using specific VGLUT1 promoter fragments (Zhang et al, 2011).…”

“…1). We previously developed antibody-mediated targeted gene transfer to specific types of neurons with HSV-1 vectors (Cao et al, 2010; Cao et al, 2011). The Staphylococcus A ZZ domain, which binds antibodies, was added to a protein on the surface of an HSV-1 vector particle, glycoprotein C (gC), to yield gC--ZZ.…”

“…Upon release, this peptide neurotransmitter binds to NMDA receptors on postsynaptic neurons. The second gene transfer selectively targeted the postsynaptic neurons in PER cortex, using antibody-mediated targeted gene transfer (Cao et al, 2010; Cao et al, 2011) and a His tag antibody, as the peptide neurotransmitter contains the His tag. With untargeted gene transfer, ~3 % of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite.…”

Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow

“…Of note, the capability to match specific axons to specific cell bodies based on hue could be used to map multiple axon collaterals, in multiple neocortical areas, that arise from a single neuron. Additional specificity for the subtype of neuron that is labeled could be obtained from using specific neuron-subtype specific promoters to express Brainbow and/or from targeting gene transfer to specific types of neurons (Cao et al, 2011). Further, postsynaptic neurons could be identified by either coexpressing a transneuronal marker, such as wheat germ agglutinin (Zhang et al, 2010b), with Brainbow, or by targeted gene transfer of HSV-1 vectors across specific synapses (Zhang et al, 2012).…”