Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters

Abstract

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors.

Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats.

Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats.

Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Keywords: HSV-1 amplicon vectors; organotypic cultures; peripheral ganglia; sensory neurons; specific promoter.

Figure 1

HSV-1-based amplicon plasmids used in this study. The scheme shows the general structure of the amplicon plasmids: oriS and pac (white circle and square respectively) correspond to the origin of viral DNA replication and the packaging sequences of HSV-1, both of which are required to generate vector particles. The blue left-arrow represents the IE4/5 promoter sequence, an HSV-1 immediate-early promoter that drives expression of the RLuc-GFP fusion reporter protein. This transcription unit is terminated by the bovine growth hormone polyadenylation signal. The blue right-arrow corresponds to the different promoter candidates, or to the control E1Fα promoter to be tested, all of which drive the expression of FLuc (orange arrow). This transcription unit is terminated by the SV40 polyadenylation signal. Each amplicon plasmid also contains the gene that confers ampicillin resistance (AmpR, blue square) and an E. coli DNA replication origin (blue circle) that allows the plasmid to be amplified in bacteria. Each amplicon plasmid (and the corresponding amplicon vector) therefore carries 2 different independent transcription units: one expresses FLuc driven by a sensory neuron-selective or control promoter candidate, and the other expresses the fused RLuc-GFP protein driven by the viral IE4/5 promoter. The GFP moiety of the RLuc-GFP protein allows vector titration by counting green infected cells. The RLuc moiety of the protein serves as an internal invariable control; it compensates for differences in the number of infected neurons and can be used as a comparator to evaluate the strength of the different selective promoter candidates. Thus, based on the ratio of FLuc/RLuc, these constructs allow the assessment of both the relative strength of different promoters in a same cell type or ganglia, and the tissue selectivity of a given promoter in different ganglia.

Figure 2

Validation of adult rat organotypic dorsal root ganglia (DRG) cultures. (a) Western blot to detect NeuN and GAPDH expression in organotypic DRG cultures at day 0, day 5 and 7 (D0, D5, D7), from control (CTL) and SCI rats (n = 2) + is the positive control (rat’s brain). (b) Neuronal survival in DRG cultures prepared from spinalized (SCI) and non-spinalized control (CTL) rats assessed by semiquantitative Western blot, with NeuN expression normalized with respect to GAPDH; initial neuronal death (up to day 5) was followed by stability (from days 5 to 7 (n = 2)). (c) Neurite outgrowth in representative DRG cultures from 2 to 7 days of culture (D2, D5, D7). Arrows indicate neurites growth after 7 days of cultures. (d) Neurite length and density (manually calculated) increased significantly from day 5 (D5) to day 7 (D7). (e) CGRP release by the adult rat organotypic DRG cultures at 3 days of culture, with (release post stimulation) or without (basal release) stimulation with 75 mM of KCl (n = 8) for 30 min. The Student t test was performed to assess differences between conditions: * p < 0.05, and *** p < 0.0001.

Figure 3

Transduction of adult rat organotypic dorsal root ganglion (DRG) cultures by HSV-1 amplicon vectors. (a) Fluc expression 24 h after infection with 10⁶ vector particles (n = 4, blank OD was 450). The background FLuc expression was 450 OD. Fluc expression increased significantly from day 3: Fluc quantifications for data at day 1 and 2 were significantly different from each value at day 3, 4 and 5. The Student t test was performed to assess differences in transgene expression between these ganglia; ns: not significant; *: p < 0.05. (b) Transduction of DRG S1 neurons with HSV-1 amplicons expressing GFP. Ganglia at day 4 of culture were analyzed by fluorescence microscopy 24 h after infection (24 hpi). OD: optical density.

Figure 4

Organotypic neuronal cultures of adult rat dorsal root ganglia (DRG), superior cervical ganglia (SCG), and paracervical ganglia (PCG) were infected after 3 days in culture with 2.10⁶ vector particles, and protein extracts were prepared after 24 h for analysis of luciferase activity. The histogram bars show normalized ratios (FLuc/RLuc, n = 3 per promoter and per ganglion type), which quantify the activity of Fluc driven by a candidate promoter relative to the activity of RLuc (in the RLuc-GFP fusion protein), which is driven by the constant ubiquitous HSV-1 IE4/5 promoter. The ANOVA test was performed to assess differences in transgene expression between these ganglia; ns: not significant; ** p < 0.001. Of the candidate promoters, only rCGRP appeared to be selective for DRG sensory neurons. The control EF1α promoter showed no selectivity, as expected. OD: optical density.

Figure 5

Organotypic neuronal cultures of adult rat dorsal root ganglia (DRG), superior cervical ganglia (SCG), and paracervical ganglia (PCG) were infected after 3 days in culture with 1.10⁶ particles of vector expressing FLuc driven by the TRPV-1 promoter, and protein extracts were prepared after 24 h for analysis of luciferase activity. The histogram bars show normalized ratios (FLuc/RLuc, n = 3 per promoter and per ganglion type), which quantify the activity of Fluc driven by a candidate promoter relative to the activity of RLuc (in the RLuc-GFP fusion protein), which is driven by the constant ubiquitous HSV-1 IE4/5 promoter. Results are presented as percentages of DRG values, considered to be 100%. Data from control rats are those previously shown in Figure 4. The ANOVA test was performed to assess differences in transgene expression between these ganglia, ns: not significant; ** p < 0.001. In organotypic cultures from SCI rats, the TRPV-1 promoter exhibited significant selective activity compared to that in autonomous ganglia that innervate the bladder. OD: optical density. IMG: inferior mesenteric ganglion.