Next-generation replication-defective HSV vectors for delivery of large DNA payloads

Figures

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  Figure 1. HSV-1 life cycle, virus structure, and replication (A) Diagrammatic representation of HSV infection in the PNS shows viral particles binding to axonal processes, followed by fusion of the viral envelope with the neuronal membrane. Viral capsid proteins then engage molecular motors for retrograde axonal transport to the neuronal cell body, where the viral genome is injected into the nucleus through nuclear pores. There, the virus either enters latency or undergoes lytic infection, producing new viral particles that travel back down the axon via anterograde transport and are eventually released from the neuronal termini. (B) Diagram and electron micrograph of an HSV particle showing the 152-kbp dsDNA genome enclosed within the icosahedral-shaped nucleocapsid, surrounded by the tegument region and an envelope that contains the viral glycoproteins, including those involved in virus attachment and entry (gB, gC, gD, and gH/gL). (C) Wild-type (WT) HSV infection proceeds through a complicated cascade of gene expression, where the tegument protein VP16 that migrates with the particle activates expression of the viral immediate-early (IE) genes, including the ICP4 and ICP27 essential…

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  Figure 2. Structure of HSV WT genome and replication-defective HSV-derived vectors (A) The HSV genome consists of two segments, unique long (UL) and unique short (US), separated by the reiterated JOINT region. IE genes (ICP0, ICP4, ICP22, ICP47, and ICP27) are shown in white boxes. HSV endogenous insulator sequences are marked by colored asterisks, indicating their positions. (B) NP2 is an rdHSV vector with deletions of ICP27 and ICP4 (Δ, black boxes). The promoter element for ICP22 and ICP47 has been deleted (ΔpICP22, ΔpICP47). Two copies of the human preproenkephalin gene are inserted in the deleted ICP4 loci, under a CMV promoter. The transgene located in the JOINT is flanked by the ICP4 insulator CTRS3 (red square) upstream, while the downstream insulator CTRS1/2 is deleted. The second transgene copy in the terminal repeat is flanked by the ICP4 insulator elements CTRS3 upstream (red square) and CTRS1/2 downstream (pink square). (C) B-VEC is an rdHSV vector deleted for ICP4 and ICP22 (Δ, black boxes). Two full-length copies of the human collagen type VII alpha 1 chain (COL7A1) gene, each with independent expression controls, are inserted into each ICP4 locus. The specific insertion…

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  Figure 3. Impact of cellular insulators in the ICP4 locus on in vivo transgene expression (A) Luciferase activity (fLuc) expressed as bioluminescence signals (p/s) upon JΔNI vector delivery to mouse hippocampus. Black circle, background bioluminescence from animals’ fur; light gray circle, fLuc activity from vector with expression cassette within the ICP4 locus (viral insulator; n = 3) out to 6 months post-vector delivery; orange circle, fLuc activity from 2tR-SMAR-supplemented expression cassette within the ICP4 locus (viral/cellular insulators; n = 3) out to 18 months. Data are means ± SEMs per group. (B) Representative coronal section of the mouse hippocampus injected with 2tR-SMAR-supplemented expression cassette within the ICP4 (viral/cellular insulators) at 6 (top) and 18 (bottom) months post-vector injection (mpi). ZsGreen+ (ZsG+) cells in green, NeuroTrace (NT) staining of neuronal body in red, and ZsG and NT merged image. Horizontal bar, 250 μm.

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  Figure 4. Schematic of cGMP production and purification of rdHSV vectors deleted for ICP4, ICP27, and ICP0 We previously engineered a U2OS cell line that expresses both HSV IE genes ICP4 and ICP27 in trans since the U2OS line can complement ICP0 mutants. We developed a protocol to propagate these cells in T-flasks and multilayer cell factories to seed small-scale and eventually large-scale bioreactors. We have determined the proper multiplicity of infection (MOI) to provide optimal virus growth and production in serum-free media (VP-SFM). After 1 day at 37°C, we shifted the infected cultures to 33°C. This temperature shift created an optimal environment for virus replication while slowing cell division, which enhanced virus spread. To increase the release of virus from the infected cells and remaining cell debris, we introduced both dextran sulfate and sodium chloride to lyse open remaining virus-infected cells and keep released virus from binding back to heparin sulfate residues on portions of the lysed cells. The released virus in the supernatant was harvested and processed using tangential flow filtration (TFF) to concentrate the virus and adjust the solution for salt-active nuclease…