Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo

Abstract

Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604-5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

FIG. 1

Viruses used in this study. (a) Vector backbones. In each case, the indicated genes have been deleted or inactivated. Further details are given in Materials and Methods. (b) Promoter cassettes. The three promoter cassettes used in the study and their insertion sites into the viral genome are indicated. Details are given in Materials and Methods.

FIG. 2

Virus strain 1764 27− 4− does not express significant amounts of any of the IE genes. Extracts were prepared from noncomplementing BHK cells harvested 24 h postinfection with the viruses 17+ 27− pR19GFP, 1764 27− pR20.5, and 1764 27− 4− pR20.5 over a range of MOIs. Complementing cells were infected at an MOI of 1 as a positive control. Western blots were probed for ICP0, ICP22, or ICP47.

FIG. 3

Virus strain 1764 27− 4− pR20.5 can direct the simultaneous high-level expression of two exogenous genes on complementing and noncomplementing cells. (a) Complementing cell line 27/12/M:4 (62) was infected with virus 1764 27− 4− pR20.5 at an MOI of 0.01. The plaque was photographed under fluorescence 72 h after infection, stained for lacZ expression, and photographed again. (b) Duplicate wells of noncomplementing BHK cells were infected with virus 1764 27− 4− pR20.5 at an MOI of 1. Forty-eight hours postinfection, one well was stained with X-Gal, and then the wells were photographed under bright field (for lacZ) or fluorescence (for GFP).

FIG. 4

Expression of exogenous genes from virus 1764 27− is not dose dependent. Noncomplementing BHK cells were infected at MOIs from 0.01 to 10 with virus strains 17+ 27− pR19GFP, 1764 27− pR20.5, and 1764 27− 4− pR20.5. Complementing 27/12/M:4 cells (62) were infected at an MOI of 1 as a positive control (indicated as + in the figure). In the case of the 17+ 27− pR19GFP virus, the positive control was run on a separate gel and is not shown. All samples were harvested at 48 h postinfection.

FIG. 5

ICP6 expression from 17+ 27−, 1764 27−, and 1764 27− 4− viruses. Western blots of extracts from BHK cells prepared 48 h after infection with virus strains 17+ 27− pR19GFP, 1764 27− pR20.5, and 1764 27− 4− pR20.5 at MOIs of 10, 5, and 1 were probed with an anti-ICP6 antibody. 27/12/M:4 cells (62) infected with each of the viruses at an MOI of 1 are shown as a positive control.

FIG. 6

Virus strain 1764 27− 4− is not toxic to primary neurons in culture. DRG neurons from an adult rat were either mock infected or infected at an MOI of 10 with virus strain 17+ 27− pR19GFP or virus strain 1764 27− 4− pR20.5. One week postinfection, cells were photographed under phase-contrast and fluorescence optics. Cells infected with the 1764 27− 4− virus (GFP and phase contrast) and mock-infected cells (phase contrast only) are also shown at higher magnification (* and **, respectively).

FIG. 7

Virus strain 1764 27− 4− is persistently maintained in cultured cells. Vero cells at 50% confluency were infected at an MOI of 10 with virus strain 1764 27− 4− pR20.5. The cells were maintained in 2% serum at 34°C. At the last time point, a duplicate well was superinfected at an MOI of 5 with virus strain 17+ 27− (expressing no reporter gene). GFP expression from 1764 27− 4− pR20.5-infected cells is shown at 3, 7, and 23 days after infection without superinfection and after superinfection at 23 days.

FIG. 8

Transgene expression from virus strain 1764 27− 4− pR20.5 is maintained for at least 3 weeks in organotypic hippocampal slice cultures. Organotypic hippocampal slice cultures were prepared and infected 7 days later with 10⁶ PFU of 1764 27− 4− pR20.5. An overview of an infected slice stained for lacZ and expression of GFP at 2, 7, and 21 days after infection are shown. The insert shows a higher-magnification image of an infected neuron at 21 days after infection.

FIG. 9

1764 27− 4− pR19lacZ is transported from the injection site in the CNS, giving widespread gene delivery. Adult Lewis rats were stereotaxically injected with 2.5 × 10⁵ PFU of 1764 27− 4− pR19lacZ either in the striatum (injection 1), at the C6 level of the spinal cord (injection 2), or in the superior colliculus and thalamus (injection 3). One week (injection 1) or 3 weeks (injections 2 and 3) after injection, the animals were perfusion fixed, and relevant areas of the nervous system were sectioned and stained for lacZ expression. The sections from injection 1 are 200 μm thick, and the sections from injections 2 and 3 are 80 μm thick.

FIG. 10

Transgene expression from differently disabled viruses containing the pR19lacZ expression cassette in the rat CNS in vivo. Either 1764 27− 4− pR19lacZ, 1764 27− pR19lacZ, or 17+ 27− pR19lacZ (2.5 × 10⁵ PFU) was stereotaxically injected into the rat striatum. Animals were perfusion fixed, and 200-μm sections were cut in the parasagittal plane to visualize the striatum and substantia nigra in the same section. (a) lacZ expression at 3 days after injection. (b) lacZ expression at 1 week after injection. (c) lacZ expression at 1 month after injection. The left panel shows transgene expression following injection of the 17+ 27− pR19lacZ virus, the middle panel shows the 1764 27− pR19lacZ virus, and the right panel shows the 1764 27− 4− pR19lacZ virus, as indicated at the bottom of the figure.