Non-toxic HSV vectors for efficient gene delivery applications and complementary cells for their production

Abstract

The present invention provides herpes simplex virus (HSV) vectors that do not express toxic HSV genes in non-complementing cells and comprise a genome containing one or more transgenes, wherein the vector is capable of expressing the transgene in non-complementing cells for at least 28 days. The vectors disclosed herein include vectors having deletions, or alternative inactivating mutations, in the genes ICP0, ICP4, ICP22, ICP27, and ICP47, or vectors that express one or more of these genes under conditions of altered kinetics. The present invention also relates to viral reservoirs of the vectors of the present invention, compositions suitable for therapeutic use or for in vitro application, and methods related thereto. On the other hand, the present invention provides complementing cells, particularly U20S cells, that are engineered to express ICP4 and ICP27 when the cells are infected with HSV, for use in producing the vectors of the present invention. The cells are disclosed as naturally complementing ICPO.

Description

[0001] This application is a divisional application of the invention patent application with application date of July 17, 2014, application number 201480051273.7, and invention name “Non-toxic HSV vector for effective gene delivery applications and supplementary cells for its production”.

[0002] Cross-reference to related applications.

[0003] This application claims priority to U.S. Provisional Patent Application No. 61 / 847,405, filed July 17, 2013, the entire contents of which are incorporated herein in their entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] This invention was made with government support under Grant Nos. PO1DK044935 and 5RO1NS064988 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0006] Among the many viral and non-viral genetic vector systems, vectors based on herpes simplex virus (HSV) have been studied for use as gene transfer vectors, including for possible therapeutic use in human patients. HSV is a complex, non-integrating DNA virus that can infect a wide range of human and animal cells. The viral genome contains more than 80 genes and consists of two distinct parts, the U and the L and U S The HSV virus is composed of two subunits, each flanked by inverted repeats encoding key diploid genes. An important feature of HSV replication is the expression of its genes in batches, known as cascade regulation (Rajcani, Virus Genes, 28: 293-310 (2004)). Removal of the key immediate early (IE) genes ICP27 and ICP4 renders the virus completely defective and unable to express the early (E) genes involved in viral genome replication and the late (L) genes whose function is in the assembly of progeny viral particles. These replication-deficient viruses can grow on complementing cells that express the (complemented) deleted ICP4 and ICP27 gene products and can subsequently be used to infect non-complementing cells, where the viral genome persists as a stable nuclear episome. However, vectors that retain the ICP0 and ICP22 IE genes are toxic to cells, but inactivating or deleting these genes, especially the ICP0 gene, hinders transgene expression.

[0007] Therefore, there remains a need for HSV vectors that are capable of expressing transgenes in any tissue or cell, in vitro or in vivo, without damaging the cell or tissue, and systems for propagating such vectors. SUMMARY OF THE INVENTION

[0008] The present invention relates to a breakthrough in the modification of HSV vectors, which provides the possibility of expressing transgenes in a variety of tissues or cells (particularly mammalian) in vitro or in vivo without expressing any harmful viral genes. The HSV vectors of the present invention do not express any toxic viral genes in non-complementing cells, but are still able to express transgenes robustly and continuously (e.g., for at least 14 days, such as at least 28 days, and preferably at least 60 days). Therefore, it fills an extremely important gap in vector technology, as HSV is the only vector that combines the ability to carry large single or multiple transgene expression cassettes controlled by universal or cell-specific promoters with efficient infectious properties in the absence of vector integration. The vectors of the present invention will allow efficient gene delivery to tissues, such as the liver, for which no effective vectors are currently available.

[0009] In one embodiment, the present invention provides a herpes simplex virus (HSV) vector that does not express toxic HSV genes in non-complementing cells and comprises a genome comprising one or more transgenes, wherein the vector is capable of expressing the transgene in non-complementing cells for at least 28 days. The vector of the present invention may comprise a transgene inserted into the genome in operably linked relation to one or more insulator sequences, wherein the vector does not express ICP0, ICP4, ICP22, ICP27, and ICP47, which are immediate early genes. Depending on the promoter controlling the activity of the transgene, the vector of the present invention can express the transgene in any type of mammalian (especially human) cell that it can infect without the cytotoxicity associated with viral gene expression.

[0010] In another aspect, the present invention provides complementing cells for producing the vectors of the present invention.The cell line of the present invention is derived from U2OS cells that have been engineered to express ICP4 and ICP27 when the cells are infected with HSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A is a schematic diagram of a vector showing reporter gene expression. The top line represents the full-length wild-type HSV-1 strain KOS BAC clone (Gierash, J. Virol. Meth., 135:197-206 (2006)) used to generate the JΔNI vector of the present invention. The second line represents the backbone of JΔNI5, i.e., JΔNI7-GFP. The third line is a magnification of the LAT to UL4 region of JΔNI5. The left side of the fourth line (LAT:CAG-GFP) shows the location of the reporter expression cassette in JΔNI7-GFP. The right side of the fourth line (UL3 / 4:CAG-GFP) shows the location of the same expression cassette in a control vector derived from JΔNI5 (JΔNI6-CAGGFP). Figure 1 A Symbol list: TR, terminal repeat; IR, internal repeat; UL, unique long region; US, unique short; numbers indicate the location of different IE genes; Δ, deletion; β, early promoter; CTRL, insulator; LAT P2, long-term expression element; CAG, CMV / actin / globin enhancer / promoter / intron cassette. Figure 1 B is a set of photographs showing reporter gene expression. Figure 1 Table B: HDF, human dermal fibroblasts; hpi, hours post infection; dpi, days post infection; MOI, multiplicity of infection.

[0012] Figures 2A-2B A set of photographs comparing transgene expression in cells infected with JΔNI6-CAGGFP and JΔNI7-GFP. Figure 2A Figure 2B shows the duration of transgene expression in infected HDFs (MOI = 0.5). Images were obtained 1-14 days after infection.

[0013] Figure 3 A set of photographs showing the expansion of JΔNI7-miR302GFP virus on complemented (U2OS-ICP4 / ICP27) cells. The photographs were taken 3 days after infection.

[0014] Figure 4 Schematic diagram of the construction of a targeting plasmid for inserting a tetracycline-inducible promoter and a Gateway recombination cassette into the LAT locus of an HSV vector. Figure 4 Symbols: Zeo, bleomycin-resistance gene; Cm, chloramphenicol-resistance gene; ccdB, toxin gene for negative selection; LATP2, LAT long-term expression element; CTRL2, chromatin boundary / insulator element 2 of the LAT locus.

[0015] Figure 5 Schematic diagram of the construction of the lentiviral plasmid for expressing ICP27 in the U2OS-ICP4 cell line. Figure 5 Symbols: p, promoter; bla, blasticidin-resistance gene.

[0016] Figure 6A is a set of photographs showing ICP27 immunofluorescence staining of U2OS-ICP4 cells and different clones of U2OS-ICP4 / ICP27 cells infected with QOZHG virus (ICP4-null; ICP27-null). Figure 6B is a set of photographs showing the growth of QOZHG virus (GFP expression from an HCMV promoter-GFP cassette in the viral genome) in U2OS-ICP4 and U2OS-ICP4 / ICP27 cells.

[0017] Figure 7 Schematic diagram of the HSV vector of the present invention (JΔNI7-GFP, middle), the LAT region of JΔNI7-GFP (top), and the LAT region of wild-type HSV (bottom).

[0018] Figure 8 This is the sequence of the LAT region of the JΔNI7-GFP HSV vector of the present invention.

[0019] Figure 9 A-9C. Vector genome construction and complementing cells for virus production. Figure 9A) Schematic representation of the wild-type HSV-1 KOS genome in the KOS-37 BAC (24) and the genomes of its IE gene-deleted derivatives JΔNI2, JΔNI3, and JΔNI5. UL, unique long segment; US, unique short segment. Open frame: terminal and internal inverted repeats. BAC elements, including the chloramphenicol-resistance gene and the β-galactosidase expression cassette, are located between loxP sites in the UL37-UL38 intergenic region (Gierasch et al., J. Virol. Methods 135, 197-206 (2006)). The US region in the KOS-37 BAC and its derivatives is compared in reverse to a standard representation of the HSV genome. Deletions in the JΔNI construct are indicated by black boxes and Δ symbols; the ICP47 promoter and translation start codon were removed as part of the joint deletion. IE genes that were switched to early expression kinetics by promoter replacement (ICP0, ICP27) or TAATGARAT deletion (ICP22) are indicated by shaded boxes and a β symbol before the ICP number. All JΔNI recombinants contain a highly activating N / T mutation in the gB gene (Uchida et al., J. Virol. 84, 12200-09 (2010)) and an ubiquitin C promoter (UbCp)-mCherry cassette in the ICP4 locus; the SV40 polyA region of the mCherry cassette is indicated by a small patterned box. ( Figure 9 B) Western blot analysis of complemented cells. Uninfected cells and cells infected with QOZHG (left) or JΔNI5 virus (right) at an MOI of 1 were harvested at 24 hpi, and extracts were prepared for gel electrophoresis. Blots were probed with antibodies against ICP4, ICP27, or α-tubulin as a loading control. ( Figure 9 C) Growth of JΔNI2 and JΔNI5 viruses in U2OS, U2OS-ICP4, U2OS-ICP4 / 27, and Vero-7b cells. Cells were infected at an MOI of 0.001 and extracellular virus was harvested from triplicate wells daily and titered on U2OS-ICP4 / 27 cells.

[0020] Figure 10 A-10B. Relative nuclear viral DNA levels after infection with equal amounts of gc or PFU. HDFs were infected with the indicated JΔNI vectors at 5,000 gc / cell ( Figure 10 A) or 1 PFU / cell ( Figure 10 B) Infection. At 2 hpi, nuclear DNA was isolated and relative viral gc numbers were determined by qPCR of the gD gene normalized to the cellular 18S rRNA gene.

[0021] Figure 11 A-11D. JΔNI cytotoxicity and viral gene expression in non-complemented cells. ( FIG. 11A ) In vitro cytotoxicity assay. HDF and Vero cells were infected at 25,000 gc / cell and cell viability was measured by MTT assay in triplicate wells at 5 dpi. The plotted values ​​represent the mean ratio of virus-infected versus mock-infected cells. Brackets with asterisks indicate statistically significant differences (p<0.05) between JΔNI2- and JΔNI3-infected Vero cells and between JΔNI3- and JΔNI5-infected Vero cells. ( Figure 11 B). Western blot analysis of IE gene products in HDFs. Cells were infected with KOS, QOZHG, or JΔNI virus at 1 PFU / cell and extracts were prepared 24 hpi. Blots were probed with antibodies against the indicated IE gene products or α-tubulin as a loading control. Figure 11 C) JΔNI IE gene expression measured by qRT-PCR. HDFs were infected with the indicated viruses at 1,000 gc / cell. mRNA was isolated at 12 hpi and reverse transcribed for qPCR to determine cDNA levels for the genes listed at the top. Expression was normalized to 18S rRNA levels and shown relative to JΔNI2-infected cells. Figure 11 D) qRT-PCR analysis of the expression of early (upper panel) and late genes (lower panel). Figure 11 C). ICP6 can be considered a delayed IE gene and is classified as an early gene in this article because its expression is reported to be more dependent on ICPO than VP16 or ICP4 (Desai et al., J. Virol. 67, 6125-35 (1993); Sze et al., Virus Res. 26, 141-52 (1992); Harkness et al., J. Virol. 88 (12) 6847-61 (2014)).

[0022] Figure 12 A-Reporter gene expression in 12D.JΔNI-infected HDFs. ( Figure 12 A) mCherry fluorescence. Cells were infected with the indicated gc / cell and photographed at 24 hpi. ( Figure 12 B) Relative mCherry mRNA levels. HDFs were infected at 5,000 gc / cell and harvested at 6 hpi for mRNA isolation, reverse transcription, and qPCR. Figure 11 C. ( Figure 12 C) mCherry fluorescence in U2OS cells. Cells were infected at 1,000 gc / cell and photographed at 24 hpi. Figure 12D) Induction of mCherry expression in JΔNI5-infected HDFs. Cells were infected at the indicated gc / cell, superinfected with QOZHG at 5,000 gc / cell 24 h later, and photographed 24 h later.

[0023] Figure 13 A-13D. JΔNI6GFP and JΔNI7GFP genomic structure and reporter gene expression. (Figure 13A) JΔNI7GFP contains a CAG promoter-EGFP expression cassette within a 2-kb LAT intronic region between the LATP2 long-term expression / enhancer region and a downstream CTCF-binding motif (CTRL2) in the intron. LATP2 extends from the LAT transcription start site into the 2-kb intron. JΔNI6GFP contains the same CAG promoter-EGFP expression cassette between the UL3 and UL4 genes. The rabbit β-globin polyA region of the CAGp-EGFP cassette is indicated by a small patterned box. (BD) EGFP and mCherry expression in infected HDFs. ( Figure 13 B) Cells were infected with JΔNI6GFP or JΔNI7GFP virus at different gc / cell and fluorescence was visualized at 3 dpi. Figure 13 C) HDFs were infected with JΔNI6GFP or JΔNI7GFP vector at 12,500 gc / cell and harvested 3 or 5 days later for mRNA extraction and qRT-PCR analysis of two reporter genes. Expression normalized to 18S rRNA is shown relative to JΔNI6GFP-infected cells at day 3. ( Figure 13 D) HDFs were infected with JΔNI6GFP or JΔNI7GFP virus at 25,000 gc / cell and EGFP fluorescence was photographed at 7, 14, and 28 dpi.

[0024] Figure 14 A-14C. Effect of LAT locus elements on EGFP expression from JΔNI7GFP. ( Figure 14 A) Genomic representation of JΔNI7GFP and derivatives with deletions of CTRL1 (ΔC1), CTRL2 (ΔC2), or LATP2 (ΔLP2), individually or in combination. The deletion of positions 8978–9161 in JQ673480 includes CTRL1, and the deletion of positions 5694–5857 in JQ673480 includes CTRL2. These deletions include some bases outside the CTCF binding motif. Figure 14 B) Reporter gene expression in infected HDFs. Cells were infected with the indicated viruses at 12,500 gc / cell and fluorescence was recorded at 3 dpi. Figure 14C) Relative EGFP mRNA levels in HDFs infected with JΔNI7GFP, LAT element-deleted derivatives, or JΔNI6GFP; viruses are identified by abbreviated names. Cells were infected at 12,500 gc / cell and processed for qRT-PCR analysis at 3 days post-infection. Expression levels were normalized to 18S rRNA and are shown relative to levels in cells infected with JΔNI7GFP.

[0025] Figure 15 A-15D. Anti-silencing activity of LAT sequences located elsewhere in the viral genome. Figure 15 A) Construction of the JΔNI9 and JΔNI10 vectors. The XhoI fragment encompassing CTRL1, LATP2, and CTRL2 was removed from the JΔNI5 genome, and a GW recombination cassette was introduced between UL45 and UL46 to generate JΔNI9GW or between UL50 and UL51 to generate JΔNI10GW (top). The same XhoI site was used to isolate the LAT fragment containing CAGp-GFP from JΔNI7GFP (bottom left). The XhoI fragment was cloned into pENTR1A (bottom right) and transferred to JΔNI9GW or JΔNI10GW via attL / attR recombination using the respective GW cassette (LR reaction) to generate JΔNI9LAT-GFP and JΔNI10LAT-GFP, respectively. As a control, a CAGp-GFP cassette without a LAT sequence was recombined into the GW locus of JΔNI9GW or JΔNI10GW via the pENTR1A intermediate, generating JΔNI9GFP and JΔNI10GFP. Figure 15 B) Reporter gene expression in HDFs infected with JΔNI9 or JΔNI10 viruses. HDFs were infected with the indicated viruses at 12,500 gc / cell. EGFP and mCherry fluorescence were recorded at 3 dpi. Figure 15 C) EGFP mRNA levels in infected HDFs determined by qRT-PCR as in the previous figures. Levels are shown relative to those in cells infected with JΔNI9GFP or JΔNI10GFP. Similar to the construction of JΔNI10LAT-GFP described above, JΔNI10ΔC12LP2-GFP was constructed by transferring the XhoI LAT fragment from JΔNI7ΔC12LP2-GFP into the GW site of JΔNI10GW. Figure 15 D) Effect of deletion of both CTRL and LATP2 from JΔNI10LAT-GFP on transgene expression. HDFs were infected at 12,500 gc / cell and EGFP and mCherry fluorescence were recorded at 3 dpi.

[0026] Figure 16 A-16D. Reporter gene expression from the JΔNI vector in other non-complementing cells. Figure 16 A) The cells listed at the top were infected with JΔNI6GFP or JΔNI7GFP at the gc / cell indicated below the graph. EGFP and mCherry fluorescence were recorded at 3 dpi. Figure 16 B) Measured by qRT-PCR analysis at 3 dpi as in ( Figure 16 EGFP gene expression in cells infected as in A). Results normalized to 18S rRNA are shown relative to cells infected with JΔNI6GFP. ( Figure 16 C) hMDSCs were infected with JΔNI6GFP or JΔNI7GFP virus at 50,000 gc / cell and EGFP fluorescence was photographed at 14 and 28 dpi. ( Figure 16 D) qRT-PCR analysis of EGFP mRNA levels in hEK, hPAD, and hHEP cells 3 days after infection with JΔNI10GFP or JΔNI10:LAT-GFP at 12,500 gc / cell. Normalized expression is shown relative to cells infected with JΔNI10GFP.

[0027] Figure 17 The construction of JΔNI8 from JΔNI5 is shown diagrammatically.

[0028] Figure 18 Shown are data for the growth of JΔNI8 relative to JΔNI5 in complementing cells (U2OS-ICP4 / 27) following infection at 1 genome copy (gc) / cell. The upper panel shows reporter gene expression (mCherry), while the lower left panel reports viral yield in plaque forming units (PFU), and the lower right panel reports viral yield in genome copies (gc).

[0029] Figure 19 Shown are data demonstrating earlier viral gene expression from JΔNI8 compared to JΔNI5 in complemented cells. In each graph, the lower bar represents data for JΔNI5, while the upper bar represents data for JΔNI8. Data were collected by qRT-PCR and expressed as fold difference relative to the JΔNI5 data point at 6 hours post-infection (hpi).

[0030] Figure 20 The genomic structures of JDNI7GFP (also known as JΔNI7GFP) and JDNI8GFP (also known as JΔNI8GFP) are shown in a diagram.

[0031] Figure 21Shown are data demonstrating that infection of human dermal fibroblast (HDF) cells with equal amounts of JΔNI8 and JΔNI5 (denoted as gc) results in equivalent amounts of viral DNA in the nucleus. Data represent two hours post-infection (hpi).

[0032] Figure 22 Shown are data comparing cell viability (MTT) of HDFs infected with different HSV vectors at two MOIs. The lower X-line in each graph represents the results for JΔNI5, while the upper X-line in each graph represents the results for JΔNI8.

[0033] Figure 23 Shown are data from a cell viability assay (MTT) comparing HDFs infected with different HSV vectors at varying numbers of viral genome copies / cell. For the left panel (12,500 gc / cell), the MOI for JΔNI5 was 5 PFU / cell, while the MOI for JΔNI8 was 18 PFU / cell. For the right panel (25,000 gc / cell), the MOI for JΔNI5 was 11, while the MOI for JΔNI8 was 33. The lower X-line in each panel represents the results for JΔNI5, while the upper X-line in each panel represents the results for JΔNI8.

[0034] Figure 24 Data comparing the viability of cells infected with KOS, JΔNI5, and JΔNI8 in six cell types (HDFs, human neonatal keratinocytes, human neural stem cells, Vero, human preadipocytes, and human hepatocytes). MTT assays were performed using 25,000 gc / cell and data are reported 5 days post infection (dpi).

[0035] Figure 25 Shown are dose response data comparing reporter gene expression (mCherry or enhanced green fluorescent protein (EGFP)) between human dermal fibroblasts (HDFs) infected with JΔNI7GFP and JΔNI8GFP at the indicated gc / cell three days after infection.

[0036] Figure 26 Shown are time course data comparing reporter gene expression (mCherry or EGFP) between human dermal fibroblasts (HDFs) infected with JΔNI7GFP and JΔNI8GFP at 25,000 gc / cell.

[0037] Figure 27Shown are the results of experiments comparing reporter gene expression (mCherry or EGFP) in human neonatal keratinocytes three days after infection with 12,500 gc / cell or 25,000 gc / cell of JΔNI7GFP or JΔNI8GFP.

[0038] Figure 28 Shown are the results of experiments comparing reporter gene expression (mCherry or EGFP) in rat dorsal root ganglion (DRG) neurons three days after infection with 6250 gc / cell of JΔNI7GFP or JΔNI8GFP.

[0039] Figure 29 Shown are results from experiments investigating transgene expression (mCherry or EGFP) in neurons infected with JΔNI7GFP. Top, separate (left; 40x) or merged (center, right; 20x) images of EGFP and mCherry fluorescence at the indicated days post-infection (dpi); bottom, separate and merged images (10x) at 15 dpi.

[0040] Figure 30 Schematic diagram of pCX4Hyg-Cre

[0041] Figure 31 is a schematic representation of the generation of pCX4Hyg-Cre.

[0042] Figure 32 Shown are data comparing EGFP mRNA levels between JΔNI7GFP and JΔNI6GFP infected HDF cells as determined by quantitative reverse transcription (RT)-PCR. Detailed Description of the Invention

[0043] The following patents and publications related to various HSV vector technologies are incorporated herein by reference. U.S. Patent No. 5,658,724 relates to HSV strains deficient in ICP4 and ICP27 and methods for their production, growth, and use. U.S. Patent No. 5,804,413 relates to cell lines containing DNA encoding ICP4, ICP27, and ICP0. U.S. Patent No. 5,849,571 relates to latently active herpes virus promoters and their uses. U.S. Patent No. 5,849,572 relates to HSV-1 vectors containing the LAT promoter. U.S. Patent No. 5,879,934 relates to recombinant HSV vectors containing genomic mutations within the ICP4 and ICP27 genes that render the ICP4 and ICP27 gene products deficient. U.S. Patent No. 5,998,174 relates to methods for preparing HSV vectors. U.S. Patent No. 6,261,552 relates to an HSV vector comprising an HSV genome having a deletion or mutation within the natural TAATGARAT sequence, wherein the deletion or mutation results in a delay in the expression kinetics of the natural immediate early genes within the genome when the genome is in a cell containing the HSV ICP4 gene product. U.S. Patent No. 7,078,029 relates to an HSV genome having a mutation of the TAATGARAT sequence such that, in the presence of the ICP4 gene product, the natural immediate early genes are expressed from the genome with delayed kinetics. U.S. Patent No. 7,531,167 relates to an HSV vector comprising a deletion only in the ICP4, ICP27, and UL55 genes. U.S. Patent Application Publication No. 2013 / 0096186 relates to an HSV vector comprising a mutant gB and / or mutant gH glycoprotein. International Patent Application Publication No. WO 1999 / 06583 relates to an HSV comprising an envelope comprising a non-natural ligand.

[0044] In one embodiment, the present invention provides a herpes simplex virus (HSV) vector that does not express toxic natural HSV genes in non-complementing cells and can continuously express a transgenic. For example, the HSV vector of the present invention can express a transgenic in cultured human dermal fibroblasts (HDF) cells for at least 14 days, such as at least 28 days, and preferably at least 60 days. It is desirable that the expression of a transgenic from the vector of the present invention occurs in the absence of a measurable ICPO gene product in such cells, such as in the absence of any ICPO gene product in such cells. In a particular embodiment, a vector according to the present invention having a transgenic (e.g., encoding enhanced green fluorescent protein (EGFP) or other marker) in a LAT region inserted between a LAT P2 element and a CTRL2 element and having both CTRL1 and CTRL2 can be determined by quantitative RT-PCR seven days after infection to have another identical genetic mutation but wherein the transgenic is inserted between the LAT P2 element and the CTRL2 element.L 3 and U L 4 is capable of expressing the transgene at least 20 (or at least about 20) times the level of the vector and at least 30 (or at least about 30) times the level as determined by quantitative RT-PCR three days after infection. Figure 13 and 32 Desirably, the vector expresses the transgene at the level and for the time period described, wherein the transgene is under the operable control of the CMV enhancer / chicken β-actin promoter / chimeric intron. The cells can be cultured for the time period described while infected with the HSV vector of the invention, which is also evidence that the vector is non-toxic to the cells (i.e., does not express toxic native HSV genes in the cells). Of course, the non-complementing cells can also be another cell type, and can be in vivo cells intended for therapeutic applications.

[0045] For example, an HSV vector of the present invention may comprise a genome comprising a transgene inserted (a) within the latency associated transcript (LAT) gene region, (b) within the ICP4 locus (and preferably only one, wherein the junction is deleted, as discussed herein) and / or (c) within the genome of the vector, operably linked to one or more insulator sequences within the genome. Preferably, the vector does not express ICP0, ICP4, ICP22, ICP27, and ICP47 as immediate early genes (although in some embodiments, expression of ICP47 may be desirable). Without wishing to be bound by theory, it is believed that, depending on the activity of the promoter within the transgene, the vector of the present invention can express the transgene in any type of mammalian (particularly human) cell that it can infect without the cytotoxicity associated with viral gene expression. The vector of the present invention can be present as isolated DNA, intracellular DNA, or packaged within a viral envelope.

[0046] Any suitable method that makes the vector of the present invention unable to express ICP0, ICP4, ICP22, ICP27 and ICP47 as immediate early genes in non-complementing cells can be used. For example, the genome of the vector of the present invention can be transformed into an inactivation mutation (e.g., deletion) comprising one or all of these genes (e.g., deletion within the coding sequence of one or more of the ICP0, ICP4, ICP22, ICP27 and ICP47 genes or the entire coding sequence (preferably comprising at least ICP0, ICP4 and ICP27 inactivation deletions, more preferably comprising ICP0, ICP4, ICP27 and ICP47 inactivation deletions), or alternatively comprising the promoter or other regulatory sequences of the genes). Alternatively, one or more of these HSV genes can be transformed into being expressed as early or late genes. For example, the genome of certain embodiments of the vector of the present invention can be transformed into retaining one or more of these genes in the coding sequence, but replacing its promoter with a promoter that makes the gene expressed as an early (β) or late (γ) rather than an immediate early (α) gene. For example, such a gene can be placed under the control of a promoter that responds to ICP4 (preferably, at least with respect to ICP22, such that ICP22 is expressed as an early gene rather than an immediate early gene). A suitable promoter for expressing such a gene with early (β) kinetics is the HSV tk promoter. The ICP22 promoter can be converted to early kinetics by truncation, i.e., deletion of regulatory sequences (including TAATGARAT). The entire ICP47 promoter and start codon can be deleted. Alternatively, in some embodiments, the ICP47 gene can be expressed as an immediate early gene to protect infected cells from immune recognition (Hill et al., Nature 1995, 375(6530):411-415; Goldsmith et al., J Exp Med. 1998; 187(3):341-348).

[0047] In addition to the interference of ICPO, ICP4, ICP22, ICP27 and ICP47 expression, it is desirable that the vector of the present invention also does not express UL41 (i.e., host shutoff (vhs) gene). UL41 is an RNase that degrades many host and viral mRNAs, causing rapid shutoff of host cell protein synthesis, and enters cells as a component of the virion envelope. Thus, for example, the gene encoding UL41 can be deleted from the genome of the vector of the present invention. Without wishing to be bound by theory, it is believed that this operation further enhances the ability of the vector of the present invention to grow in supplemented cells by saving the ICP4 and ICP27 mRNAs that are supplemented, and enhances the expression of the transgene in non-supplemented cells by saving the transgene mRNA.

[0048] It should be recognized that the genomic sequences of several HSV strains are known to those skilled in the art (e.g., MacDonald, J. Virol, 86(11): 6371 (2012); McGeoch, J. Gen. Virol., 69: 1531-1574 (1988); GenBank Accession No. JQ673480; NCBI Reference Sequence: NC_001806.1; MacDonald, J. Virol. 86(17): 9540 (2012); GenBank Accession No. JX142173, which are incorporated herein by reference). Thus, manipulation of the sequences of HSV genes and loci is within the ordinary skill level. It should also be noted that these disclosed sequences are merely exemplary, and that other HSV strains or variants can be used as source genomes in engineering the vectors of the present invention.

[0049] In addition, the genome of the vector of the present invention can include a bacterial artificial chromosome (BAC) box. Including the BAC box promotes the proliferation and operation of the genome of the vector of the present invention in bacteria. The BAC box can include sequences that contribute to the bacterial expression of bacterial strains, for example, optional genes, such as genes that confer resistance to antibiotics or toxins (for example, preferably chloramphenicol, but other resistance genes (for example, tetracycline, ampicillin, bleomycin resistance genes) can also be used). The BAC box can also be included in a eukaryotic promoter, such as a reporter gene under the control of a constitutive mammalian promoter (for example, SV40, RSV, CMV, ubiquitin C (UbC), CAG, or β-actin promoter, etc.), or a gene encoding a fluorescent protein (for example, g (encoding green fluorescent protein), y (encoding yellow fluorescent protein), r (encoding red fluorescent protein), and its analogs (for example, encoding iRFP, EGFP, etc.)).

[0050] The BAC cassette can be placed in the genome of the vector of the present invention at any suitable location, such as the UL37-UL38 intergenic region within the vector genome (e.g., Gierash, J. Virol. Meth., 135: 197-206 (2006) and Morimoto, Microbiol. Immunol., 53: 155-161 (2009)). In addition, it is desirable that the BAC cassette be flanked by sequences that facilitate removal of the BAC cassette, such as by site-specific recombinase recognition sites / consensus sequences (e.g., those recognized by enzymes such as cre, dre, flp, KD, B2, B3, R, etc.). Because BAC sequences have been shown to reduce viral growth in cultured cells (e.g., Gierash, J. Virol. Meth., 135: 197-206 (2006)), including such sites facilitates excision of the BAC cassette, if desired. In addition, excision of the BAC cassette can increase the ability of the vector to integrate one or more transgenes, since the BAC cassette is approximately 11 kb. It will be understood that the vectors of the invention may also have a consensus sequence for a recombinase (e.g., loxP), particularly a consensus sequence that is not native to the HSV genome, for example as a result of removing the BAC cassette using cells expressing an appropriate site-specific recombinase for excising the BAC cassette, thereby leaving a single copy of the recombinase consensus sequence within the HSV genome (e.g., within the UL37-UL38 intergenic region, if this is where the BAC cassette was inserted).

[0051] As described above, the vectors of the present invention can include at least one transgene inserted into the HSV vector genome in operably linked to one or more insulator sequences. By "operably linked," it is understood that the one or more insulator sequences allow the transgene to be expressed in a cellular environment where genetic elements (i.e., "genes") otherwise present within the HSV genome are transcriptionally silenced. Without wishing to be bound by any particular theory, it is believed that such insulator sequences prevent the formation of heterochromatin (which, if formed, silences gene expression) at the site of such insulator sequence and within about 1 kb to about 5 kb thereof. Thus, the insulator sequences used in the vectors of the present invention are generally sequences that interfere with the binding or formation of heterochromatin, which would otherwise silence the expression of the transgene. Non-limiting examples of suitable insulator sequences include the HSV chromatin boundary (CTRL / CTCF-binding / insulator) elements CTRL1 and CTRL2 (which may be native to the LAT locus, as described herein, or moved to an ectopic location within the genome), the chicken hypersensitive site 4 insulator (cHS4), the human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), and the scaffold / matrix attachment region (S / MAR) from the human interferon beta gene (IFNB1) (Emery, Hum. Gene Ther. 22, 761-74 (2011); Antoniou et al., Hum. Gene Ther. 24, 363-74 (2013)).

[0052] In addition to being inserted into the vicinity of natural insulator sequences (such as CTRL1 and CTRL2 sequences in the LAT region) for the HSV genome, insulator sequences can be inserted into the vector genome at any suitable site. These insulator / boundary elements can be introduced into the genome of the vector of the present invention by standard methods, and can be included in the box identical with the transgenic, or introduced into the genome respectively, so that the flank or otherwise are operably connected to a given transgenic cassette. Therefore, it is possible to insert such a gene cassette, which includes, for example, one or more ectopic insulator sequences that are similar in function to those naturally found on the transgenic flank in the LAT region or that are operably connected to transgenic insulator sequences in other ways. It will be understood that vectors of the present invention can include multiple transgenics that are operably connected to multiple insulator sequences (included at the abnormal site relative to LAT). In embodiments where one or more transgenes are inserted at a site other than the LAT and are operably linked to CTRL1 and / or CTRL2, it is desirable to delete or mutate the CTRL1 and / or CTRL2 sequences within the LAT to minimize or eliminate recombination events between native sequences within the LAT of the vectors of the invention and those engineered to be operably linked to the transgene within an ectopic (non-LAT) site. When CTRL1 and CTRL2 remain within the LAT or are moved ectopically, a preferred site for insertion of the transgene into the genome of the vectors of the invention is between CTRL1 and CTRL2 (e.g., within approximately 1-4 kb of each of CTRL1 and CTRL2, with CTRL1 and CTRL2 flanking the transgene).

[0053] In the vectors of the present invention, one or more of the insulator sequences are operably linked to the transgene so that the transgene is isolated from gene silencing and expressed. Typically, the transgene and insulator sequences should be adjacent to each other within the genome, such as separated by less than about 5 kb, or less than about 4 kb, or less than about 3 kb, or less than about 2 kb, or less than about 1 kb. It may also be desirable for the expression cassette (including the transgene) to be functionally between the two insulator sequences so that the insulator sequence flanks the transgene under investigation (see, e.g., Emery, Hum. Gene Ther. 22, 761-74 (2011); Antoniou et al., Hum. Gene Ther. 24, 363-74 (2013)).

[0054] One preferred site for inserting a transgene (e.g., a first transgene) in the vectors of the present invention is between insulator sequences within the LAT gene region of the vector genome—specifically, between chromatin boundary (CTRL / CTCF-binding / insulator) elements located upstream (CTRL1) of the LAT promoter LAP1 and within the LAT 2-kb intron (CTRL2), respectively (Amelio et al., J Virol 2006, 80(5):2358-2368; Bloom, Biochim. Biophys. Acta, 1799:246-256 (2010)). This region is referred to herein as the LAT (gene) region or locus. Thus, it is desirable that the genome of the vectors of the present invention comprise (e.g., retain) CTRL1 and CTRL2 (see Figure 7 , top). Without wishing to be bound by theory, it is believed that the presence of CTRL1 and CTRL2 protects the region from forming heterochromatin and thus contributes to the LAT gene region becoming a specialized site for expressing transgenes. Thus, the vector expresses transgenes inserted into the LAT gene region in non-complementing cells. In a preferred embodiment, the vector comprises multiple transgene cassettes within the LAT gene region, each comprising a separate promoter and coding region, and each of which can be mono- or polycistronic.

[0055] In addition, it is preferred for the LAT region of the vector of the present invention (which comprises at least one transgene, as described herein) to also comprise (e.g., retain) a LATP2 or LAP2 enhancer element. Again, without wishing to be bound by theory, it is believed that the presence of the LATP2 or LAP2 enhancer element contributes to the ability of the transgene within the LAT gene region to express the coding sequence for a long period of time (Goins, J. Virol., 73:519-532 (1999); Lilley, J. Virol., 75:4343-4356 (2001)). In a particularly preferred embodiment, the transgene within the LAT gene region is inserted downstream of the LATP2 or LAP2 enhancer element. However, the present invention contemplates embodiments in which the transgene is inserted upstream of the LATP2 or LAP2 enhancer element (relative to the direction of LAT transcription). Desirably, the transgene is distal to the LATP2 or LAP2 element within the LAT gene region (see, e.g., Figure 7 ,top).

[0056] Another preferred site for inserting a transgene (e.g., a second transgene) in the vectors of the present invention is within the ICP4 locus. For example, a transgene controlled by the UbC promoter inserted into this locus of the vectors of the present invention can generate long-term signals in hippocampal neurons and be at least short-term active in DRG.

[0057] The transgene can also be inserted into the genome of the vector of the present invention in operable association with (e.g., in proximity (<5 kb)) other insulator sequences. In addition to being inserted near the insulator sequence native to the HSV genome, the insulator sequence can be inserted into the vector genome at any suitable site. Thus, it is possible to insert a gene cassette that includes, for example, ectopic insulator sequences that are functionally similar to those naturally found flanking the transgene in the LAT region. It will be understood that the vector of the present invention can include multiple transgenes.

[0058] In the transgene inserted into the vector of the present invention, there is at least one promoter sequence and a transcribed sequence such that the transcribed sequence is controlled by the promoter. The promoter in the transgene can be any promoter required to control / regulate the expression of the transcribed sequence. For example, the promoter can be a cell-specific or tissue-specific promoter (e.g., EOS, OCT4, Nanog (for ESC / iPSC), SOX2 (for neural stem cells), αMHC, Brachyury, Tau, GFAP, NSE, synapsin I (for neurons), ApoA-I, albumin, ApoE (for liver), MCK, SMC α-actin, myosin heavy chain, myosin light chain (for muscle) etc.), such as a promoter that specifically or preferentially expresses a gene in a defined cell type (e.g., in hepatocytes, lung cells, epithelial cells, heart cells, neural cells, skeletal muscle cells, embryonic, induced pluripotent or other stem cells, cancer cells, etc.). Preferred promoters for sensory neurons include TRPV1, CGRP and NF200. In other embodiments, the promoter within the transgene inserted into the vector of the present invention can be an inducible promoter (e.g., TRE3G in combination with rtTA3G expression, separated from a promoter in LAT or other inducible promoters known in the art). Of course, the promoter within the transgene expression cassette inserted into the vector of the present invention can be a constitutive mammalian promoter, such as those known in the art (e.g., SV40, CMV, CAG, EF1α, UbC, RSV, β-actin, PGK, etc.).

[0059] In addition to promoter and coding sequence, the transgenic in the genome of the vector of the present invention can also include other regulatory elements.For example, the transgenic can include one or more sites for combining microRNA.In a preferred embodiment, the transgenic comprises the binding site for the microRNA of series connection, such as 2,3,4,5 or 6 sites (four are typical) of series connection. The site is particularly used for the existence of the series connection binding site of the microRNA, promotes the expression of the transgenic in some cell types. Therefore, for example, the vector comprising the transgenic (which can be toxic for a lot of cell types) that hopes to express in cancer or tumor cells can include the binding site for the microRNA of "normal" (i.e. non-malignant) cell, so that the expression of the transgenic is suppressed in non-malignant cells.

[0060] It should be noted that the transgene within the vectors of the present invention can be monocistronic (i.e., encoding a single protein or polypeptide) or polycistronic (i.e., encoding multiple proteins or polypeptides). In addition, for example, all or part of the transcribed portion of the transgene can also encode a non-translated RNA, such as an siRNA or miRNA. Furthermore, the vectors of the present invention can contain multiple separate monocistronic or polycistronic transgene units (preferably two separate transgene units, but possibly more (e.g., three, four, five or more separate units)), each with its own corresponding promoter, translated sequence or non-translated RNA sequence, and other regulatory elements.

[0061] As described, a transgene comprises one or more transcribed sequences that are expressed under the control of a promoter and optionally other regulatory elements within the transgene (including being operably linked to an insulator sequence). The transcribed sequence can be any sequence that is desired to be expressed in a given cell (into which the vector is introduced). Non-limiting examples of transcribed sequences that can be present in a transgene within a vector of the invention include Oct4, Klf4, Sox2, c-Myc, L-myc, dominant negative p53, Nanog, Glis1, Lin28, TFIID, GATA4, Nkx2.5, Tbx5, Mef2C, Myocd, Hand2, SRF, Mesp1, SMARCD3, SERCA2a, Pax3, MyoD, Lhx2, FoxG1, FoxP2, Isl1, Ctip2, Tbr1, Ebf1, Gsx2, Srebp2, Factor VIII, Factor IX, Dystrophin, CFTR, GlyRα1, enkephalin, GAD67 (or other GAD isoforms, e.g., GAD 65), TNF, IL-4, neurotrophic factors (e.g., NGF, BDNF, GDNF, NT-3), Ascl1, Nurr1, Lmx1A, Brn2, Myt11, NeuroD1, FoxA2, ​​Hnf4α, Foxa1, Foxa2 or Foxa3, any microRNA or combination of miRNAs (e.g., hsa-mir-302 / 367 gene cluster; hsa-miR200c; hsa-miR369; hsa-mir-124) and / or one or more other non-coding RNAs ("ncRNAs") or reporter genes for expression in mammalian cells, such as LacZ (encoding β-galactosidase), CAT (encoding chloramphenicol acetyltransferase), or fluorescent protein encoding genes (e.g., GFP, YFP, RFP, and analogs thereof such as iRFP, EGFP, etc.).

[0062] In addition to the aforementioned, the vector of the present invention can also optionally include an expression cassette that is inserted in a site other than the LAT region or other than a known insulator sequence. The preferred site of the expression cassette is ICP4. For example, when the vector comprises the complete or inactivation deletion of the ICP4 gene, the expression cassette can be inserted in the site of ICP4 deletion. It is desirable that the coding sequence of the expression cassette inserted in the site other than the LAT region is controlled by a constitutive mammalian promoter (e.g., SV40, CMV, CAG, EF1α UbC, RSV, beta-actin, PGK, etc.), but if desired, other promoters (as discussed herein and known in the art) can be used. An exemplary expression cassette includes the expression driven by the UbCp of the mCherry constructed into the ICP4 locus of the deletion. Of course, the transgenic can also encode a factor of therapeutic interest.

[0063] In addition to the above, it is desirable that the HSV vector of the present invention further comprises an internal repeat (junction) region (which comprises the IR S and IR L ). Deletion of this region can contribute to the stability of the vector genome, and deletion of this sequence of HSV DNA also allows the vector to accommodate large transgenes (at least 15kb) and still be correctly packaged into mature virions. The deletion of the junction eliminates one copy of each of the IE genes ICP0 and ICP4, so that the remaining copies can be easily manipulated. It also deletes the promoter of the ICP22 or ICP47 immediate early gene. If necessary, the expression of the ICP47 gene can be restored by inserting an immediate early promoter, preferably the ICP0 promoter or the HCMV major IE promoter, to minimize immune recognition of infected cells (Hill et al., Nature 1995, 375 (6530): 411-415; Goldsmith et al., J Exp Med. 1998; 187 (3): 341-348).

[0064] HSV is able to infect a variety of mammalian cells; therefore, the vectors of the present invention have a wide range of applications. However, in order to enhance infectivity, it is desirable that the envelope of the vectors of the present invention may also comprise mutant glycoproteins that enhance infection and / or lateral spread relative to wild-type glycoproteins. Alternatively or in addition, the envelope of the vectors of the present invention may also comprise mutant glycoproteins that direct HSV into cells through non-canonical receptors. For example, the mutant glycoprotein may be gB, gC, gD, gH or gK; of course, the vector may have more than one of the mutant (enhanced penetration or spread) glycoproteins (e.g., a combination of two, more, or even all of them). In addition, techniques for mutating the glycoprotein to enhance HSV infection and / or lateral spread are known, and any of the techniques can be used in the context of the present invention (see, e.g., U.S. Patent Application Publication No. 2013-0096186 A1; International Patent Application Publication No. WO / 1999 / 006583, Uchida, J. Virol., 84: 12200-12209 (2010), Uchida et al., J. Virol., 87(3). 1430-42 (2013), and Uchida, Mol. Ther., 21: 561-569 (2013), which are incorporated herein by reference). In addition, the genome of the vector of the present invention may comprise a mutant gene encoding the mutant glycoprotein.

[0065] Exemplary vector "backbones" for practicing the present invention are described herein as "JΔNI5" and "JΔNI8," with the understanding that, as "backbones," it is contemplated that transgenes with or without external control elements can be inserted into the LAT region of these specific vectors (see, e.g., Figure 7 ,top).

[0066] One application of the vectors of the present invention is to reprogram a variety of cell types to generate pluripotent stem cells. In recent years, stem cells have been at the forefront of biomedical research and offer much hope for understanding human development, genetic diseases, and generating new therapeutic approaches in regenerative medicine. In 2006, Yamanaka and colleagues discovered a method for generating embryonic-like stem cells by reprogramming adult human fibroblasts (Takahashi and Yamanaka, Cell 126:663-76, 2006). These new cells, designated induced pluripotent stem (iPS) cells, have similar functions to ES cells (Wemig et al., Nature 448:318-24, 2007) and, when derived from human somatic cells (Takahashi et al., Cell 131:861-72, 2007; Yu et al., Science 318:1917-20, 2007), circumvent the ethical issues associated with the use of human ES cells. Initially, four reprogramming genes were used for iPS cell generation (Takahashi and Yamanaka, Cell 126:663-76, 2006; Takahashi et al., Cell 131:861-72, 2007), but the efficiency of reprogramming with these and other genes remained problematic due to inefficient gene transfer methods. The vector systems of the present invention address this problem by combining high transduction efficiency for many cell types with the ability to simultaneously express multiple transgenes from a single vector. For example, the JΔNI7 and JΔNI8 vectors described herein are replication-defective and non-toxic due to the deletion or altered expression kinetics of the five viral IE genes. Because they do not integrate into the cellular genome, the vectors are diluted during cell division, providing a hit-and-run gene delivery system.

[0067] To facilitate the cultivation, production, and amplification of the vectors of the present invention and the production of their stocks, one aspect of the present invention provides a complementing cell line that complements ICPO and ICP4, desirably ICPO, ICP4, and ICP27. Preferably, ICPO complementation is achieved without expressing HSV ICPO to reduce toxicity within the complementing cells. Therefore, preferred complementing cells according to the present invention are derived from cell types that naturally complement HSV ICPO function, such as U2OS cells (Yao, J. Virol., 69: 6249-6258 (1995), which is incorporated herein by reference). The cells can be engineered to express ICP4 or ICP4 and ICP27 by methods known in the art (e.g., by introducing an ICP4 or ICP4 and ICP27 expression cassette into the cells so that they express ICP4 and ICP27, respectively, from a genetic construct rather than the HSV genome, such as from a cell chromosome). Desirably, the cell line expresses ICP4 and ICP27, respectively, in trans. Desirably, the introduced ICP4 or ICP4 / ICP27 coding sequence is under the control of its cognate viral promoter. Additionally, expression of one or both of the ICP4 and ICP27 supplementary coding sequences can be induced in the cell in response to HSV infection.

[0068] As mentioned above, the embodiment of the carrier of the present invention comprises BAC, and the flank of described BAC is the sequence that promotes the removal of BAC box by site-specific recombinase recognition.Therefore, the supplementary cell of the present invention can be transformed into the gene of the site-specific recombinase that further expresses the recognition sequence that coding is suitable for in the carrier, thereby produces recombinase protein.Therefore, the supplementary cell of the present invention can express and produce cre, dre, flp, KD, or B2, B3, R etc., or its mutant derivative as appropriate.Therefore, make transgenic (about 11kb) regain space by described cell line, and can make virus growth improve more than 10 times, as more than 25 times, or more than 50 times, as about 100 times.Compared with the cell lacking described recombinase, the virus growth improved can be measured (producing growth curve) by standard procedure.This relates to with low MOI infected cell multiple hole, virus collection at different time after infection, and conventionally by the yield titration of plaque assay.

[0069] In addition, the complementing cell line can be engineered to express a gene encoding a selectable marker, such as markers commonly used to engineer packaging cells or cells expressing any other exogenous gene. Suitable selectable genes include those that confer resistance to neomycin / G418, hygromycin, blasticidin, puromycin, bleomycin, and the like.

[0070] It will be appreciated that methods for engineering a source cell type (e.g., U2OS cells) to contain expression constructs encoding HSV ICP4 and ICP27 proteins and other proteins (e.g., recombinases and / or selectable gene products) are known to those of ordinary skill in the art. For example, the gene of interest with a selectable marker can be subcloned into a lentiviral vector, the source cells infected with the lentiviral vector, selection for marker expression (e.g., blasticidin resistance), and then confirmation of expression of the transgene of interest (e.g., HSV ICP27).

[0071] Of course, the complementing cells of the present invention can be propagated and cloned. Thus, the present invention provides clonal populations, ie, cell lines, comprising, consisting of, or consisting essentially of the complementing cell lines as described herein.

[0072] Using the complementing cells of the present invention, the HSV vectors of the present invention can be propagated. Therefore, the present invention provides a method for propagating the HSV vectors of the present invention. According to the method of the present invention, the complementing cell line is transfected with the vector DNA and then cultured until plaques form. The viral DNA as described can have a BAC, and, if so, the cells of the present invention can express a recombinase suitable for excising the BAC from the viral genome (if it is desired not to include a BAC in the packaged vector). The viral population is amplified by repeatedly transferring the infectious particles to an increasing number of large, fresh complementing cell populations. For these repeated transfers, the multiplicity of infection (MOI) can be between about 0.001 pfu / cell and about 0.03 pfu / cell. Finally, the vector of the present invention (as packaged virus) is purified from cells with a 90% cytopathic effect.

[0073] In general, the HSV vectors of the invention are most useful when enough virus can be delivered to a cell population to ensure that the cells are exposed to an appropriate amount of virus. Accordingly, the invention provides stocks, preferably homologous stocks, comprising the HSV vectors of the invention. The preparation and analysis of HSV stocks are well known in the art. For example, viral stocks can be made in roller bottles containing cells transduced with an HSV vector. The viral stock can then be purified on a continuous nycodenze gradient and divided into aliquots and stored until needed. Viral stocks vary greatly in titer, which depends primarily on the viral genotype and the protocol and cell line used to prepare them. Preferably, such stocks have a titer of about 10 6 pfu / ml or even more preferably about 10 7 In a more preferred embodiment, the titer may be about 10 pfu / ml. 8 pfu / ml, or approximately 109 pfu / ml (or at least about that value), and about 10 10 pfu / ml or about 10 11 pfu / ml or even about 10 12 High titer stocks of 10 pfu / ml (or at least about said value) are most preferred. Thus, the titer of an HSV stock according to the present invention may be from about 10 6 pfu / ml is about 10 12 pfu / ml (preferably about 10 9 to about 10 11 pfu / ml). The genome copy number (gc) provides a cell line-independent measure of the number of viral particles, but includes defective particles. Typically, the gc value of wild-type HSV-1 is several to 20 times, up to 100 times, higher than the pfu value of the same stock. For mutant viruses, particularly defective viruses grown on complementing cells, this can increase to as much as 10,000 times or even more. Gc and pfu values ​​increase proportionally with the size of the stock.

[0074] In addition, the present invention provides a composition comprising an HSV vector and a carrier, preferably a physiological carrier. The carrier of the composition can be any carrier suitable for the vector. The carrier is typically a liquid, but can also be a solid, or a combination of liquid and solid components. Ideally, the carrier is a pharmaceutical (e.g., physiologically or pharmacologically acceptable) carrier (e.g., an excipient or diluent). Pharmaceutical carriers are well known and readily available. The choice of carrier will be at least partially determined by the specific vector and the specific method for administering the composition. The composition may also include any other suitable ingredients, particularly ingredients for enhancing the stability of the composition and / or its end use. Therefore, there are a variety of suitable formulations for the compositions of the present invention. The following formulations and methods are exemplary only and are not intended to be limiting in any way.

[0075] The preparation that is suitable for parenteral administration comprises aqueous and non-aqueous isotonic sterile injection solution, it can contain antioxidant, buffer, antibacterial agent, and makes the solute that preparation and the blood of expected recipient are isotonic, and can comprise the aqueous and non-aqueous sterile suspension of the following: suspending agent, solubilizing agent, thickening agent, stabilizing agent and antiseptic.Preparation can be with the container of unit dose or multi-dose sealing, As ampoule and bottle exist, and can store under freeze drying (lyophilizing) condition, only need to add sterile liquid excipient such as water for injection at once before use.Interim injection solution and suspension can be prepared from sterile powder, granule and the tablet of the kind described before.

[0076] In addition, the composition may contain additional therapeutic or bioactive agents. For example, therapeutic factors for treating specific indications may be present. Factors that control inflammation, such as ibuprofen or steroids, may be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector, as well as physiological pain. Immune system suppressants may be administered with the composition method to reduce any immune response to the vector itself or to the disease. Alternatively, immunopotentiators may be included in the composition to upregulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, may be present to reduce the risk of infection associated with the gene transfer procedure and other conditions.

[0077] Using the vector of the present invention (and the storage and composition comprising the vector), the present invention provides a method for expressing a transgene in a nucleated cell, particularly a non-complementary cell. According to this method, the vector of the present invention is exposed to the cell under conditions suitable for vector-infected cells. Once the cell is infected, the transgene inserted in the LAT region of the vector will be transcribed (expressed) in the cell, provided that the promoter in the transgene is an active promoter in the cell and that the transgene is not suppressed by another regulatory mechanism (e.g., microRNA discussed herein). In other words, the vector of the present invention serves as a gene transfer and expression vector in mammalian cells.

[0078] As desired, the methods of the present invention can be used to express transgenes in cells in vivo or in vitro. For in vivo use, the cells can be any desired cell type, such as exocrine secretory cells (e.g., glandular cells, such as salivary gland cells, mammary gland cells, sweat gland cells, digestive gland cells, etc.), hormone-secreting gland cells (e.g., pituitary cells, thyroid cells, parathyroid cells, adrenal gland cells, etc.), ectoderm-derived cells (e.g., keratinized epithelial cells (e.g., constituting skin and hair), wet stratified barrier epithelial cells (e.g., cornea, tongue, oral cavity, gastrointestinal tract, urethra, vagina, etc.), cells of the nervous system (e.g., peripheral and central neurons, glia, etc.), mesoderm-derived cells, cells of many internal organs (e.g., kidney, liver, pancreas, heart, lung), bone marrow cells, and tumor or other cancer cells. Preferred, non-limiting examples of cells suitable for infection with the vectors of the present invention include hepatocytes, lung cells, epithelial cells, heart cells, muscle cells, stem cells, and cancer cells.

[0079] It will be observed that when used in vivo, the methods of the present invention can treat a disease or condition in a subject when the transgene within the vector encodes one or more prophylactically or therapeutically active proteins, polypeptides, or other factors (e.g., non-coding RNA (ncRNA) such as siRNA or miRNA). Thus, the present invention provides methods of treating a disease or condition in a subject, comprising administering to the subject a vector of the present invention in an amount sufficient to infect the subject's cells, and at a location sufficient to infect the subject's cells, such that the transgene is expressed within the subject's cells, and wherein the transgene encodes one or more prophylactically or therapeutically active proteins, polypeptides, or ncRNAs. For example, the disease or condition can be a type of cancer, wherein the transgene can encode an agent that enhances tumoricidal activity (e.g., TRAIL or tumor necrosis factor (TNF)). As additional non-limiting examples, the transgene can encode an agent suitable for treating conditions such as muscular dystrophy (suitable transgenes encode dystrophin), cardiovascular disease (suitable transgenes include, for example, SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (suitable transgenes include, for example, NGF, BDNF, GDNF, NT-3, etc.), chronic pain (suitable transgenes encode GlyRα1, enkephalin, or glutamate decarboxylase (e.g., GAD65, GAD67, or another isoform), lung disease (e.g., CFTR), or hemophilia (suitable transgenes encode, for example, Factor VIII or Factor IX).

[0080] In other embodiments, the method of the present invention can be used in vitro to cause transgenic expression in cells in culture. In addition, any cell type can be infected in vitro with the method of the present invention, such as stem cells and fibroblasts, such as human dermal fibroblasts (HDF) or human lung fibroblasts (HLF). Other preferred cell types for use in vitro include keratinocytes, peripheral blood mononuclear cells, hematopoietic stem cells (CD34+), or mesenchymal stem / precursor cells. In one embodiment, transgenic encoding one or more factors that can affect the differentiation of cells. For example, Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant negative p53, Nanog, Glis1, Lin28, TFIID, mir-302 / 367, or the expression of one or more of other miRNAs can cause cells to become induced pluripotent stem (iPS) cells. See also, Takahashi and Yamanaka, Cell, 126:663-676 (2006); Takahashi, Cell, 131:861-872 (2007); Wernig, Nature, 448:318-324 (2007); and Yu, Science, 318:1917-1920 (2007), the disclosures of which are incorporated herein by reference. Alternatively, the transgene within the vector of the present invention can encode one or more factors for transdifferentiating cells (e.g., GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mesp1, SMARCD3 (for cardiomyocytes); Ascl1, Nurr1, Lmx1A, Bm2, Myt11, NeuroD1, FoxA2 (for neural cells), Hnf4, Foxa1, Foxa2 or Foxa3 (for hepatocytes).

[0081] In practicing the methods of the present invention involving infecting cells in vitro or in vivo with the vectors, compositions, or reservoirs of the present invention, the cells can be any mammalian nucleated cell in which it is desired to express a transgene. HSV has a wide range of infectivity, and, as described herein, the vectors of the present invention can be engineered to alter its natural tropism and enhance infectivity by mutating the viral envelope glycoproteins. Thus, the vectors can be used to infect cells of many mammalian species. It is believed that the methods of the present invention can be applied in agriculture, such as for expressing foreign genes or for supplementing defective genes in animals such as cattle, horses, sheep, goats, pigs, etc. Similarly, the methods of the present invention can be used in the context of veterinary medicine for companion animals such as cats, dogs, etc.

[0082] Of course, the methods of the present invention can also be used in vivo in humans to provide expression of prophylactic or therapeutically active agents or factors in a medical setting. The factors (supplemented by expressing one or more transgenes within the vectors of the present invention) can be exogenous, or factors that complement genetic defects.

[0083] The following examples further illustrate the invention but, of course, should not be considered as limiting its scope in any way.

[0084] Example 1

[0085] This example describes the development of a complementing cell line for the replication and production of the HSV vectors of the present invention.

[0086] Some immediate early (IE) genes of HSV are critical for viral replication, but these and other IE genes have toxic effects in a variety of cell types. When these genes are removed to prevent vector toxicity, key products must be provided to produce infectious viral particles. A new cell line based on U2OS human osteosarcoma cells was modified to conditionally express the key IE genes ICP4 and ICP27. These genes were introduced under the control of their cognate viral promoters by retroviral-mediated insertion. Thus, these genes can remain silent until HSV infection delivers the HSV envelope protein VP16 to the nucleus, where it promotes high-level expression of the integrated ICP4 and ICP27 genes by activating their promoters. Therefore, before HSV infection, these genes can be stably maintained in the modified U2OS cells without undue toxicity. HSV growth also depends on the expression of ICP0, but this protein inhibits cell replication, causing cell cycle arrest and programmed cell death. Significantly, U2OS cells naturally complement ICPO function, and therefore, the introduction of ICP4 and ICP27 is sufficient to provide a cellular environment for the efficient production of vectors lacking all three IE genes. This novel engineered cell line is stable, grows well in culture, and can be used for clinical production of non-toxic HSV vectors.

[0087] U2OS-ICP4 / 27 cells and 7b (Vero-ICP4 / ICP27) cells were infected with E1 G6 (ΔICP4::HCMVp-eGFP / ΔICP27 / β22 / β47) or JDQOZEH1 (ΔICP4 / ΔICP27 / ΔICP22 / ΔICP0::HCMV-eGFP) viruses. Three days later, the virus in the cell supernatant was titered on U2OS-ICP4 / 27 cells.

[0088]

[0089] The data showed that E1G6 (deficient in ICP4 and ICP27; no expression of ICP22 and ICP47) grew on both cell lines, whereas JDQOZEH1 (deficient in ICP0, ICP4, ICP27, and ICP22) grew only on U2OS-ICP4 / 27 cells. The JDQOZEH1 titer in the 7b supernatant likely represents residual input from the 7b infection.

[0090] Example 2

[0091] This example describes an embodiment of an HSV vector comprising a genome comprising a transgene inserted into the LAT gene region, wherein the vector does not express ICPO, ICP4, ICP22, ICP27, and ICP47 as immediate early genes.

[0092] Vector genome ( Figure 1 A) A bacterial artificial chromosome (BAC) cassette containing a Cre-deletable region in the UL37-UL38 intergenic region allows for propagation and manipulation in bacteria. L and U S The non-critical internal repeat region (junction, 14kb) is partially separated to provide space for transgenes to be inserted and to increase vector stability. Due to the deletion of ICP4 and ICP27 genes, the vector is replication-defective and, in addition, lacks the promoter and start codon of the toxic IE gene ICP0 and the ICP47 IE gene; the remaining toxic IE gene, ICP22, is controlled by the ICP4-dependent (early, β) promoter, thereby expressing ICP22 during the "early" stage of HSV gene expression in ICP4-complemented cells rather than the "immediate early" stage. Because U2OS cells naturally complement ICP0, the vector grows in the U2OS-based virus production cell line U2OS-ICP4 / ICP27 (which complements the functions of all these IE genes except the non-essential ICP47 gene). The vector genome also contains a pair of mutations in the gB gene that enhance viral entry into cells and a ubiquitin (UbC) promoter-mCherry reporter gene expression cassette at the position of ICP4 deletion. In non-complementing cells, the latency-associated transcript (LAT) promoter region, which is located between insulator (CTRL) elements that protect this region from heterochromatin formation, remains active. Furthermore, this region contains the enhancer element LATP2, or LAP2, which promotes long-term gene expression. A CAG promoter-GFP expression cassette was inserted between LATP2 and CTRL2, and robust GFP expression was observed in infected human dermal fibroblast (HDF) cells, whereas minimal expression was observed from the same GFP cassette inserted elsewhere in the genome or from the mCherry cassette. Figure 1B) Thus, in the complete absence of IE gene expression, the LAT locus is the preferred site for transgene expression.

[0093] Figure 1 A shows the structure of the complete HSV-1 genome with BAC sequences in the UL (top), and the basic vector construct. The LAT and UL3-UL4 regions are magnified below, and alternative positions for CAG-GFP insertion are indicated. Figure 1 B shows GFP expression from an alternative location in the vector genome in infected HDFs. The GFP gene within the vector encodes EGFP.

[0094] Example 3

[0095] This example lists the structure and properties of different HSV vector constructs.

[0096]

[0097] + Δ = deletion; β = conversion to early expression kinetics; Δp = promoter and start codon deletion

[0098] 1 CAG promoter

[0099] 2 Contains the first intron of EF1α after the EF1α promoter

[0100] *All have the same mCherry expression construct in the deleted ICP4 locus

[0101] **In HDF (human dermal fibroblasts), at 3 dpi

[0102] Example 4

[0103] This example demonstrates transgene expression in cells infected with JΔNI7-GFP and JΔNI6-CAGGFP. The location of CAG-GFP in JΔNI7-GFP is shown in Figure 1 In the figure, it is LAT:CAG-GFP; its position in JΔNI6-CAGGFP is shown in Figure 1 In the example, UL3 / 4:CAG-GFP is shown. In addition, the sequence of the LAT region of JΔNI7-GFP HSV is shown in Figure 8 SEQ ID NO: 1, including the sequences of the different genetic elements within the LAT region. It will be noted that the GFP within these vectors encodes EGFP.

[0104] The biological titer of the viral stock was determined on U2OS-ICP4 / ICP27 cells, and the genome copy (gc) titer was determined by quantitative real-time PCR targeting the viral glycoprotein D gene. The particle (gc) to plaque forming unit (PFU) ratios for JΔNI6-CAGGFP and JΔNI7-GFP were comparable. See Example 8, Table 2.

[0105] Non-supplementing human dermal fibroblasts (HDFs) and ICP0-supplementing U2OS cells were infected with each virus to compare their transgene expression. JΔNI7-GFP, containing a CAG-GFP cassette in the LAT locus, showed strong, viral dose-dependent GFP expression in HDFs, whereas the highest dose of JΔNI6-CAGGFP yielded only minimal GFP expression ( Figure 2A , left panel, EGFP). GFP expression in JΔNI7-GFP-infected HDFs was still detectable 2 weeks after infection ( Figure 2B , EGFP). However, little or no mCherry expression was observed from either virus in HDFs ( Figure 2A , 2B, mCherry), suggesting that genes outside the LAT locus are silenced in HDFs. In contrast, abundant GFP and mCherry expression was observed for both viruses at low MOI in U2OS cells ( Figure 2A , right column), consistent with the interpretation that the ICPO-like activity of these cells prevents silencing of non-LAT loci present in HDFs. Taken together, these results strongly suggest that the LAT locus is a preferred site for expressing transgenes from replication-defective ICPO-deficient vectors.

[0106] Example 5

[0107] This example demonstrates the production of two isolates of JΔNI7-miR302GFP BAC in U2OS-ICP4 / ICP27 cells.

[0108] The JΔNI7-miR302GFP BAC construct carries an expression cassette for the miR302s / 367 cluster in the LAT locus (Anokye-Danso, Cell Stem Cell, 8: 376-388 (2011)) instead of the typical Yamanaka somatic cell reprogramming gene cocktail (OKSM: Oct4, Klf4, Sox2, c-Myc) (Takahashi and Yamanaka, Cell, 126: 663-676 (2006); Takahashi, Cell, 131: 861-872 (2007)). The miR302s / 367 gene cluster is located in an intron connecting the EF1α promoter to the GFP coding sequence. Purification of JΔNI7-miR302GFP BAC Two isolates of DNA were prepared and introduced into U2OS-ICP4 / ICP27 cells to generate virions for examination of viral growth and transgene expression. When a 90% cytopathic effect was observed in the culture, virus was collected from the cells and supernatant and used to infect fresh U2OS-ICP4 / ICP27 cells. Transgene expression and viral spread were subsequently monitored daily. Figure 3 As shown in , three days after infection, both BAC isolates produced plaque-forming viruses and both viruses expressed EGFP and mCherry.

[0109] Example 6

[0110] This example describes the construction of a targeting plasmid for inserting a tetracycline-inducible promoter and a Gateway recombination cassette into the LAT locus of an HSV vector.

[0111] Different strategies can be used to prevent genetic rearrangement and inactivation of the OKSM expression cassette during viral expansion, where the cassette is inserted into the LAT locus of JΔNI5 or JΔNI8. One of these strategies is to replace the constitutively active CAG promoter of the OKSM cassette with a tetracycline-inducible promoter. Because the tetracycline-inducible promoter is only active in the presence of both its transactivator (rtTA) and tetracycline / doxycycline, transgene expression can be tightly regulated (repressed) during viral expansion.

[0112] A targeting plasmid was developed for insertion of a tetracycline-inducible promoter into the LAT locus ( Figure 4). A lentiviral construct (pLenti-CMVTRE3G-NeoDEST, Addgene) carrying a tetracycline-inducible TRE3G promoter was used as a starting construct. The TRE3G promoter was isolated from pLenti-CMVTRE3G-NeoDEST as a DraI-SpeI fragment and inserted between the NruI and SpeI sites of the plasmid pCMV-GW, replacing the inherent CMV promoter. The plasmid pCMV-GW contains the bleomycin (Zeo)-selection gene in the Gateway (GW) box, which replaces the chloramphenicol (Cm)-selection gene in the GW of the lentiviral plasmid. The TRE3G-GW (Zeo) expression cassette was isolated and cloned into the LAT sequence in the plasmid containing a portion of the LAT locus to add a "homologous arm" to the TRE3G-GW (Zeo) box for recombination into the BAC DNA.

[0113] Example 7

[0114] This example demonstrates immunofluorescence staining and complementation assays for ICP27.

[0115] U2OS-ICP4 cells selected for resistance to blasticidin and individual clones of ICP27 lentivirally infected U2OS-ICP4 cells were infected with QOZHG virus (ΔICP4, ΔICP27::HCMV IEp-GFP, β-ICP22, β-ICP47, ΔUL41::ICPOplacZ; Chen, J. Virol., 74:10132-10141 (2000)) at an MOI of 0.5 for ICP27 immunofluorescence staining and at an MOI of 0.01 for complementation assays. For ICP27 immunofluorescence staining, cells were fixed and stained 24 hours after infection ( Figure 6A ). Clone #1 and #8 showed strong induction of ICP27 expression by infection with ICP27-deficient HSV (QOZHG). For the complementation assay, viral growth was monitored and pictures were taken at 24, 48, and 72 hours post-infection ( Figure 6B ). U2OS-ICP4 / 27 clones #1 and #8 showed the strongest ability to support the growth of QOZHG virus.

[0116] Example 8

[0117] This example demonstrates the construction and testing of various HSV vectors.

[0118] Materials and methods

[0119] cell

[0120] Human osteosarcoma U2OS cells (ATCC, Manassas, VA, USA) were grown in DMEM with 10% FBS and penicillin-streptomycin (P / S). Human neonatal dermal fibroblasts (HDF) (ATCC, PCS-201-010) and BJ human foreskin fibroblasts (ATCC, CRL-2522) were grown in DMEM with 10% embryonic stem cell-containing FBS (Invitrogen) and P / S. Vero, Vero-7b (Krisky et al., Gene Ther. 5, 517-30 (1998)), and 293T cells were cultured in DMEM with 5% FBS and P / S. Human hepatocytes (hHEP) were isolated and cultured as described (Ueki et al., Hepatology 54, 216-28 (2011); Yoshida et al., Hepatology 58, 163-75 (2013)). Human subcutaneous preadipocytes (hPAD) (PT-5020, Lonza) were cultured with PBM TM -2 basal medium (Lonza). Human muscle-derived stem / progenitor cells (hMDSCs) were cultured as described (Gao et al., Cell Transplant 22, 2393-408 (2013)). Human neonatal keratinocytes (hEK) (Invitrogen) were cultured in 4% CO2 supplemented with human keratinocyte growth supplement. Culture medium (both from Invitrogen). Dorsal root ganglia (rDRG) were microdissected from 15-day-old rat embryos, dissociated with 3 mg / ml type I collagenase (Sigma, St. Louis, MO) in Leibovitz's L-15 medium at 37°C for 30 min with continuous shaking, and plated on poly-D-lysine (Sigma)-coated coverslips. Approximately 105 cells / well were plated in 24-well plates in 500 μl of Neurobasal medium (Gibco / Invitrogen, Grand Island, NY) supplemented with 100 ng / ml 7.0S NGF (Sigma) and a defined composition of B27 supplement, Glutamax-I, Albumax-II, and P / S. 1-3 days after plating, the cultures were treated with 10 μM uridine and 10 μM fluorodeoxyuridine (Sigma) in the above medium for 1-2 days to limit the expansion of dividing cells such as fibroblasts and glia. The cells were then washed with PBS and incubated with Neurobasal medium supplemented with NGF as above. Viral infection was performed 10-15 days after plating.

[0121] U2OS-ICP4 cells were generated by infecting U2OS cells with purified ICP4 lentivirus (see below), isolating puromycin-resistant clones (2 mg / ml), and screening for ICP4 expression by immunofluorescence after infection with an ICP4-deficient virus (QOZHG) at a multiplicity (MOI) of 0.5. Similarly, U2OS-ICP4 / 27 cells were generated by infecting U2OS-ICP4 cells with purified ICP27 lentivirus, selecting for puromycin and blasticidin (10 mg / ml) resistance, and screening QOZHG-infected clones for ICP27 immunofluorescence and virus growth ( FIG. 6 ).

[0122] Lentivirus

[0123] The lentiviral ICP4 expression plasmid pCDH-ICP4-puro was constructed by replacing the complete HCMV IE promoter of the plasmid pCDH-CMV-MCS-EF1-Puro (Systembio) with the ICP4 promoter and the coding region of plasmid S3 consisting of the SphI fragment of pICP4-lox-pac (Rasty et al., J. Neurovirol. 3, 247-64 (1997)) inserted into the SphI site of pUC19 (GenBank JQ673480.1 HSV-1 KOS map position 131, 587-124, 379; D. Krisky and JCG, unpublished). TM The blasticidin-resistance cassette of -DEST (Invitrogen) was substituted for the puromycin-resistance cassette of pCDH-CMV-MCS-EF1-Puro to form pCDH-CMV-MCS-SV40-bla to construct the lentiviral ICP27 expression plasmid pCDH-ICP27-SV40-bla ( Figure 5 The ICP27 promoter and coding region were then isolated by digesting plasmid pD7 containing the ICP27 gene and flanking sequences between the EcoRV and Sad sites (GenBank JQ673480.1 map positions 110, 580-115, 666; D. Krisky and JCG, unpublished) with BamHI (map positions 113, 244) and Sad, and the isolated fragment was used to replace the CMV promoter in pCDH-CMV-MCS-SV40-bla. The resulting plasmid, pCDH-ICP27-SV40-b1a, was cloned in Figure 5 described in .

[0124] Using ViraPower TMLentivirus packaging mix (Invitrogen) was used to produce lentivirus according to the manufacturer's instructions. Briefly, 293T cells were transfected with ViraPower TM The mixture was transfected with either pCDH-ICP4-puro or pCDH-ICP27-SV40-bla. After 2 days, the supernatant was harvested, clarified, filtered through a 0.45 μm filter, and concentrated by centrifugation. Lentivirus generated from pCDH-ICP4-puro is referred to herein as ICP4 lentivirus; lentivirus generated from pCDH-ICP27-SV40-bla is referred to herein as ICP27 lentivirus.

[0125] HSV-BAC Retrofit

[0126] All HSV-BAC constructs generated in this study and converted to virions are listed in Table 2 and were derived from KOS-37 BAC (24), a gift from D. Leib (Dartmouth Medical School, NH). All BAC modifications were made by scarless Red recombination in E. coli strain GS1783 (from G. Smith, Northwestern University, Chicago, IL) using pRed / ET (Gene Bridges, Heidelberg, Germany) and pBAD-I-sceI plasmids (kindly provided by N. Osterrieder, Free University of Berlin, Germany) or as described (Tischer et al., Methods Mol. Biol. 634, 421-30 (2010); Tischer et al., Biotechniques, 40, 191-97 (2006)), or by in vitro Gateway (GW) recombination according to the Gateway Technology Manual (Invitrogen) (http: / / tools.lifetechnologies.com / content / sfs / manuals / gatewayman.pdf). All constructs were verified by PCR analysis, FIGE analysis of restriction enzyme digestion, and targeted DNA sequencing. Targeting plasmids for Red recombination were constructed as described (Tischer et al., Biotechniques, 40, 191-97 (2006)). A kanamycin-resistance gene flanked by I-SceI restriction sites (I-SceI-aphAI fragment) was PCR amplified from pEPkan-S2 (from N. Osterrieder) (Tischer et al., Biotechniques, 40, 191-97 (2006)) using the different targeting primers specified below and listed in Table 1. Primers targeting the viral genome were designed based on the sequence of HSV-1 strain-17 (GenBank JN555585.1). All targeting fragments used for Red recombination were purified using a Qiagen gel extraction kit (Qiagen) or SpinSmart nucleic acid preparation and purification columns (Denville Scientific). The nucleotide positions provided below refer to the GenBank JQ673480 sequence of HSV-1 strain KOS.

[0127] To construct the JΔNI vector, the previously described highly active N / T double mutation was introduced into the gB gene of the KOS-37 BAC (Uchida et al., J. Virol., 84, 12200-09 (2010)), and the internal repeat (junction) region was deleted. The I-SceI-aphAI fragment was cloned into the SnaBI site of the plasmid pgB1:D285N / A549T (Uchida et al., J. Virol., 84, 12200-09 (2010)). The resulting plasmid, pgB:N / T-kan, was used as a template for amplification using primers 61 and 62 (Table 1), and the product was recombined with the native gB gene of the KOS-37 BAC, followed by I-SceI-enhanced deletion of the aphAI gene in bacteria transformed with the pBAD-I-sceI plasmid. Next, nested forward primers 46, 48 and reverse primer 47 were used to amplify the I-SceI-aphAI region for Red-mediated deletion junction (GenBank JQ673480 position 117, 080-132, 466) along with the adjacent unique short (U S ) fragment, followed by the removal of the aphAI gene. S In the opposite direction, U S 12 (ICP47) gene is placed directly adjacent to the junction, and U S 1 gene near U S A 14-nt deletion outside the terminal repeat junction (GenBank JQ673480 positions 145,377-145,390) was made to remove the ICP47 translation start codon.

[0128] Replacement of the ICP4 locus in the junction-deleted gB:N / T BAC with an mCherry expression cassette was achieved as follows: Plasmid pUbC-mCherry-SV40pA was constructed by cloning the human ubiquitin C promoter (UbCp) from pBluescriptUB-Flag-mArt (a gift from H. Nakai, Oregon Health & Science University, Portland, OR), the mCherry gene from pEP-miR (Cell Biolabs, San Diego, CA), and the SV40 polyadenylation (polyA or pA) region from pEP4-EO2SCK2M-EN2L (Addgene plasmid 20924) (Yu et al., Science 324, 797-801 (2009)) into pBluescript KS+ (Stratagene). The I-SceI-aphAI fragment was then cloned into the BamHI site of pUbC-mCherry-SV40pA at the border between UbCp and mCherry to generate pUbC-mCherry-SV40pA-KAN. The insert was PCR amplified with primers 51 and 52 (Table 1) for Red-mediated recombination using the ICP4 target locus. The resulting construct was deleted for HSV-1 KOS positions 146, 113-151, 581 of the GenBank JQ673480 sequence, including the TAATGARAT motif of the ICP22 promoter.

[0129] JΔNI2 was generated by replacing the ICP0 and ICP27 IE promoters with the early (β) HSV-1 thymidine kinase (TK) promoter by PCR using nested primer pairs 81 / 82 and 83 / 84 through the TK promoter (Craft et al., Stem Cells 26, 3119-29 (2008)) in front of both the ICP0 and ICP27 coding regions in the JΔββ viral genome. The products of each reaction were cloned into pCRblunt (Invitrogen). To generate pCRBlunt-β0 and pCRBlunt-β27, I-SceI-aphAI was inserted into the BglII site of pCRBlunt-β0 and the HpaI site of pCRBlunt-β27, resulting in pCRBlunt-β0-KAN and pCRBlunt-β27-KAN, respectively. PCR amplification of the inserts using primer pairs 85 / 86 and 87 / 88, respectively, was performed for Red recombination with a junction-deleted UbCp-mCherry gB:N / T BAC. JΔNI3 was then derived from JΔNI2 by Red-mediated complete deletion of the ICP0 coding sequence using nested targeting forward primers 41 and 42 and reverse primer 43 for I-SceI-aphAI amplification. Similarly, JΔNI5 was derived from JΔNI3 by complete deletion of the ICP27 coding sequence using primer pair 44 / 45, generating a targeted I-SceI-aphAI fragment.

[0130] JΔNI6-CAGGFP was generated by inserting an EGFP expression cassette between the UL3 and UL4 genes of JΔNI5. First, the plasmid pCAG-GFP was constructed by replacing the gH gene between the CAG promoter (CMV enhancer / chicken β-actin promoter / chimeric intron) and the rabbit β-globin polyA region in the plasmid pPEP100 (a gift from P. Spear, Northwestern University) (Pertel et al., Virology 279(1), 313-24(2001)) with the EGFP gene from pEGFP-C1 (Clontech). I-SceI-aphAI was then inserted into the SnaBI site of pCAG-GFP to generate the plasmid pCAG-GFPKAN. Separately, multi-step PCR using KOS-37 BAC DNA as a starting template was performed to generate a fragment containing a new cloning site (MCS) between the UL3 and UL4 polyA regions, as follows. First, extension PCR was performed to amplify the 3'UTRs of UL3 and UL4 using primer pairs 57 / 58 and 59 / 60, respectively, which added overlapping MCS regions to each 3'UTR fragment. Both PCR products were gel-purified, and 100 ng of each was used in overlapping PCR using primers 57 and 59 to generate a continuous fragment. This product was cloned into pCRBlunt (Invitrogen), generating the plasmid pCRBluntUL3-4linker. The insert of pCAG-GFPKAN was then cloned between the AccI and PsiI sites of the MCS of pCRBluntUL3-4linker. The resulting plasmid was digested with MfeI and PpuMI, and the UL3-CAG-GFPKAN-UL4 fragment was isolated and used for recombination with the UL3-UL4 intergenic region of JΔNI5, followed by removal of the aphAI gene.

[0131] To generate JΔNI7GFP, an XhoI fragment (~6.2 kb) containing the two CTRLs, LAP1, and LATP2, of the HSV-1 LAT locus was isolated from KOS-37 BAC DNA and cloned into pBluescript KS+. From this recombinant, an internal KpnI-SalI fragment extending from near the end of LATP2 to ~250 bp downstream of CTRL2 was isolated and cloned between the KpnI and SalI sites of pSP72 (Promega) to generate pSP72KOS-LAT. A multiple cloning site was then introduced between two BstXI sites located ~240 to ~430 bp downstream of the KpnI site, generating the pSP72KOS-LATlinker. Separately, the plasmid pCAG-GW was constructed by replacing the gH gene of pPEP100 (Pertel et al., Virology 279 (1), 313-24 (2001)) with a PCR-amplified modified GW recombination cassette [GW-Zeo; bleomycin resistance instead of chloramphenicol resistance (Wolfe et al., J. Virol. 84, 7360-68 (2010)). The insert of pCAG-GW was then cloned into the MCS of pSP72KOS-LATlinker to generate pSP72KOS-LATlinker-GW. The plasmid was digested with KpnI and HpaI to isolate the CAG-GW region with flanking LAT sequences for Red-mediated recombination with the LAT locus of JΔNI5 in ccdB-resistant Herpes Hogs bacteria (Wolfe et al., J. Virol. 84, 7360-68 (2010)). Finally, the GW cassette in the resulting JΔNI5 recombinant BAC was replaced with the EGFP gene, including the CAG and polyA sequences of the plasmid pCAG-GFPKAN, by Red-mediated recombination using an AatII-PsiI fragment. JΔNI7GFP derivatives lacking specific LAT region elements (CTRL1, CTRL2, LATP2) outside the EGFP cassette were generated by Red-mediated recombination of the JΔNI7GFP DNA with a targeted I-SceI-aphAI cassette generated by PCR using the F1, F2, and R primers listed in Table 1 (63-65, 66-68, and 69-71, respectively).

[0132] For the construction of the different JΔNI9 and JΔNI10 vectors, the ~6.2-kb LATXhoI fragment described above was deleted from the JΔNI5 genome by recombination with LAT-targeted I-SceI-aphAI generated by PCR using primers 78, 79, and 80, generating JΔNI5ΔL. GW-Zeo was then amplified using primers targeting the intergenic region between UL45 and UL46 (74 / 75) or between UL50 and UL51 (76 / 77), and the products of each reaction were recombined with JΔNI5ΔL BAC DNA to generate JΔNI9GW and JΔNI10GW. JΔNI7GFP BAC DNA was digested with XhoI, and the ~7.2-kb CAG-GFP-containing fragment from the LAT locus was isolated and cloned into pENTR1A (pENTR-LAT-XhoI). The corresponding ~5.3-kb XhoI fragment of JΔNI7GFPΔC12LP2 was similarly isolated and cloned into pENTR1A (pENTR-LATΔ-XhoI), and finally, the insert of pCAG-GFP (see above) was transferred to pENTR1A (pENTR-CAG-GFP). In vitro LR Clonase (Invitrogen) reactions were then performed to recombine the different pENTR constructs with JΔNI10GW BAC DNA to generate JΔNI10LAT-GFP, JΔNI10C12LP2-GFP, and JΔNI10GFP, respectively. Furthermore, pENTR-LAT-XhoI and pENTR-CAG-GFP were recombined with JΔNI9GW BAC DNA to generate JΔNI9LAT-GFP and JΔNI9GFP.

[0133] KNTc was constructed by introducing the gB:N / T mutation into KOS-37 BAC as described above and the UbCp-mCherry cassette into the intergenic region between the UL3 and UL4 loci of KOS-BAC by recombination with I-SceI-aphAIRed targeted amplification using primers 53 and 54.

[0134] Virus

[0135] JΔNI BAC DNA was converted into infectious virus by transfection of U2OS-based complementing cells. DNA in 500 μl of OptiMEM (Invitrogen) was incubated with 1 μl of Lipofectamine Plus reagent (Invitrogen) at room temperature for 5 minutes, 6.25 μl of Lipofectamine LTX (Invitrogen) was added, the mixture was incubated at room temperature for 30 minutes, and then added to the cells. After incubation at 37°C for 6 hours, the transfection mixture was removed and the cells were cultured overnight at 37°C using serum-free DMEM, transferred to a 33°C incubator, and monitored for 100% cytopathic effect (CPE). The supernatant was titered and subsequently amplified by infecting subsequent larger-scale cultures at a multiplicity of infection (MOI) of 0.001 PFU / cell. KNTc infectious virus was generated by transfecting Vero cells with 3 μl of Lipofectamine Plus reagent and 9 μl of Lipofectamine LTX at 37°C for 4 hours; an MOI of 0.01 PFU / cell was used for KNTc virus amplification on Vero cells. Complemented cells used for transfection and / or virus growth were as follows: U2OS-ICP4 (JΔNI2, JΔNI3), U2OS-ICP4 / 27 (JΔNI5 and derivatives), and Vero-7b [QOZHG viruses (ΔICP4, ΔICP27::HCMV IEp-GFPβ-ICP22, β-ICP47, and ΔUL41::ICPOp-lacZ) (Chen et al., J. Virol. 74, 10132-41 (2000)). The titers of all viral stocks were determined on U2OS-ICP4 / 27 cells (Table 2). Physical titers [genome copies (gc) / ml] were determined by quantitative real-time PCR as described below. Fluorescent images of infected cells were obtained at 40x magnification using a Nikon Diaphot fluorescence microscope (Nikon, Melville, PA).

[0136] Virus growth curve

[0137] Replicate wells of Vero-7b, U2OS, U2OS-ICP4, and U2OS-ICP4 / 27 cells in 24-well plates were infected at a multiplicity of infection (MOI) of 0.001 for 2 h, treated with 0.1 M glycine (pH 3.0) for 1 min to inactivate extracellular virus, and incubated at 37°C and 5% CO. Culture media were harvested daily, and titers were determined on U2OS-ICP4 / 27 cells by standard plaque assay.

[0138] Cytotoxicity assay

[0139] 5x103 HDFs and Vero cells were seeded in 96-well plates and infected with KOS, QOZHG, or JΔNI virus at 25,000 gc / cell. Cell viability was determined by MTT assay after 5 days essentially as described (Uchida et al., J. Virol. 87, 1430-42 (2013)).

[0140] Western blotting and immunofluorescence

[0141] Cell lysate preparation and Western blotting were performed as described (Miyagawa et al., PLoS One 4, e4634 (2009)). Polyclonal rabbit anti-ICP0 antibodies were produced in our laboratory, anti-ICP27 (10-H44) was from Fitzgerald Industries International (Concord, MA), anti-ICP22 was a gift from John Blaho (Mt Sinai School of Medicine, NY), anti-ICP4 (10F1) was from Santa Cruz Biotechnology, and anti-α-tubulin (T6793) was from Sigma. Immunofluorescence using the same ICP4 and ICP27 antibodies was performed essentially as described (Uchida et al., J. Virol. 83, 2951-61 (2009)) and examined under a Nikon fluorescence microscope.

[0142] Quantitative reverse transcription-PCR (qRT-PCR) and genomic PCR

[0143] For qRT-PCR, total RNA was usually extracted using the RNeasy kit (Qiagen). cDNA was synthesized using a kit (Ambion). Real-time PCR was performed using a StepOnePlus real-time PCR system (Applied Biosystems). For small numbers of cells, Cells-to-cDNA TM The PCR amplification kit (Ambion) was used for cell lysis and reverse transcription. 18S ribosomal (r) RNA results were used to normalize the data. All qRT-PCR primers used in this study are listed in Table 1.

[0144] To determine the physical (genome copy) titer of the viral stock, 5 ml of virus was incubated with 300 U / ml of Benzonase nuclease (Sigma) at 25°C for 1 h in the presence of 2 mM MgCl2, and viral DNA was extracted by DNeasy Blood & Tissue Kit (Qiagen). The gc titer was determined by qPCR targeting the glycoprotein D (gD) gene using the gD primers (38 / 39) and probe (40) listed in Table 1. The nuclei were isolated as described (Dignam et al., Nucleic Acids Res. 11, 1475-89 (1983); Suzuki et al., BMC Res. Notes 3, 294 (2010)) by washing the cells at 2 hpi, extracting DNA using the DNeasy Blood & Tissue Kit, and determining the amount of nuclear viral DNA by qPCR targeting the gD gene as above. Ribosomal RNA control reagent (Invitrogen) measures cellular 18S ribosomal DNA levels and is used to normalize viral DNA amounts.

[0145] statistics

[0146] All values ​​are presented as mean + / - SD. Differences between pairs were analyzed by Student's t-test using Microsoft Excel 14.4.1. P values ​​below 0.05 (P < 0.05) were considered statistically significant.

[0147] result

[0148] Vector transformation and viral growth

[0149] It has been reported that the defective HSV vector, JDββ, efficiently transduces mouse embryonic stem cells (mESCs) (Craft et al., Stem Cells 26, 3119-25 (2008)) without detectable damage to the cells. JDββ is deleted from the internal repeat region ("junction") of the HSV genome and two IE genes (ICP4 and ICP22). In addition, the promoters of two other IE genes (ICP0 and ICP27) are replaced with copies of the viral thymidine kinase (TK) early (E or β) gene promoter. Non-complementing cells infected with JDββ show minimal IE gene expression, but the vector is able to efficiently express transgenes in mESCs without interfering with embryoid body formation or developmental transcriptional programs. To facilitate the use of this vector backbone for a variety of gene transfer applications, Red-mediated recombination (Tischer et al., Biotechniques 40, 191-97 (2006)) was performed in bacteria to derive the backbone, JΔNI2 ( Figure 9 A). JΔNI2 was deleted for ICP4 and binding sites, including the ICP47 promoter, contained the same ICP0 and ICP27 promoter replacement scheme as JDββ, and deleted the consensus VP16-binding (TAATGARAT) motif in the ICP22 regulatory region to shift the kinetics of ICP22 expression to that of an early gene. To visualize infection and monitor viral transcriptional activity, an mCherry reporter gene expression cassette was introduced in the position of the deleted ICP4 locus. In addition, the glycoprotein B gene was replaced with a hyperactive allele, gB:N / T (Uchida et al., J. Virol 84:12200-9 (2010)) to enhance viral entry into cells. To eliminate the possibility of low-level production of toxic ICP0 and ICP27 proteins in non-complementing cells, two derivatives of JΔNI2 were also constructed by deleting the entire coding sequence of ICP0 (JΔNI3) or ICP0 and ICP27 (JΔNI5). Figure 9 A).

[0150] To convert the different JΔNI vector constructs into infectious virions, cell lines capable of complementing ICP0, ICP4, and ICP27 were generated. Human osteosarcoma U2OS cells naturally complement ICP0 (Yao et al., J. Virol. 69, 6249-58 (1995)), eliminating the need to express this toxic protein. U2OS cells were transduced with a lentivirus carrying the ICP4 gene under the control of its own regulatory sequences, and clonal lines permanently expressing ICP4 were isolated (U2OS-ICP4; Figure 9 B). Transduction of these cells with a second lentivirus carrying the ICP27 gene and control regions (Figure 6) was then used to select a cell line, U2OS-ICP4 / 27, which, when infected with the ICP4 / ICP27-deficient virus, additionally expressed ICP27 ( Figure 9 B and Figure 6). While the ability of U2OS cells to activate the ICP4 promoter and tolerate sustained ICP4 expression in the absence of viral VP16 protein was unexpected, it is unknown whether these features are related to the natural ICPO complementation activity of these cells. DNA from the BAC constructs JΔNI2 and JΔNI3 could be converted into infectious virus by transfection into U2OS-ICP4 cells, while DNA from the JΔNI5 construct only produced infectious particles when transfected into U2OS-ICP4 / 27 cells (data not shown). Figure 9 C shows the growth of JΔNI2 and JΔNI5 viruses initially generated by transfecting U2OS-ICP4 or U2OS-ICP4 / 27 cells with JΔNI2 or JΔNI5 BAC DNA, respectively. Both viruses were unable to grow on unmodified U2OS cells or Vero-7b cells supplemented with ICP4 and ICP27 but not ICP0. JΔNI2 was able to grow on U2OS-ICP4 cells without ICP27 supplementation, while JΔNI5 required this additional supplementation activity for growth. The stability of the JΔNI5 supplementation property of U2OS-ICP4 / 27 cells was tested by plaque assay at different cell passages, and no significant trend in plaque formation efficiency was observed over at least 20 passages (Table 3). These results are consistent with the engineered IE gene modifications in these viruses and allow comparison of their biological properties.

[0151] Standardization of virus input

[0152] The relative amounts of each virus required to deliver equal amounts of viral DNA to the infected cell nucleus were evaluated. The biological and physical titers of various viral stocks were determined by standard plaque assay on U2OS-ICP4 / 27 cells and qPCR for the viral glycoprotein D gene, respectively; approximately a 3-fold difference in the ratio of genome copies (gc) to plaque forming units (PFU) was observed between JΔNI2, JΔNI3, and JΔNI5 viral stocks (Table 2). HDFs were infected with the three viruses at equal PFU or equal gc and the number of HSV genomes in the nucleus was determined by qPCR 2 hours (h) post-infection (pi). When equal gc was used for infection, the number of viral genomes in the nucleus at 2 hpi was similar between JΔNI2-, JΔNI3-, and JΔNI5-infected cells ( Figure 10 A). In contrast, infection with equal PFU resulted in a greater number of JΔNI5 genomes in the nucleus compared to JΔNI2 or JΔNI3 genomes ( Figure 10 B) Therefore, in the remainder of this study, GC numbers were used to normalize viral input.

[0153] Characterization of vector properties in non-complementing cells

[0154] We first examined the effects of infection with JΔNI2, JΔNI3, and JΔNI5 on the viability of non-complementing HDFs and Vero cells compared to wild-type KOS virus and a previous IE gene-deleted vector, QOZHG (ICP4- / ICP27- / β-ICP22 / β-ICP47; (Chen et al., J. Virol. 74, 10132-41 (2000)). Five days (d) post-infection with 25,000 gc / cell, approximately 4-5 times more viable cells remained in JΔNI-infected HDF cultures compared to KOS- or QOZHG-infected cultures, whereas smaller differences were seen between JΔNI- and QOZHG-infected Vero cells ( Figure 11A). Among the JΔNI viruses, JΔNI2 was somewhat more toxic to HDFs than JΔNI3 (p=0.0017) and 5 (p=0.0430), while toxicity to Vero cells decreased from JΔNI2 to JΔNI3 (p<0.001) to JΔNI5 (p<0.001). To correlate these findings with IE gene expression, Western blots of HDFs infected at 24 hpi were probed with antibodies against four of the five IE proteins (Figure 11B). Both JΔNI2 and JΔNI3 showed residual ICP27 expression, indicating that the TK promoter preceding the ICP27 gene in these cells is not silenced in either vector. However, no IE protein was detected in cells infected with JΔNI5, despite using 2-3 times more germline cells per cell (equivalent PFU / cell, see Table 2). The KOS and QOZHG patterns are consistent with the reported downregulation of ICPO expression by ICP4 (Douville et al., Virology 207, 107-16 (1995); Resnick et al., J. Virol. 63, 2497-503 (1989)). Using quantitative reverse transcription-PCR (qRT-PCR) analysis, ICPO mRNA was detected in JΔNI2-infected HDFs and the levels of ICP22 and ICP27 mRNA were reduced between JΔNI2-, JΔNI3-, and JΔNI5-infected cells 12 h after infection with equal gc / cell ( Figure 11 C). These results suggest that JΔNI2 produces sufficient levels of ICPO to enhance ICP22 and ICP27 expression and confirm that stepwise deletion of IE genes reduces HSV vector cytotoxicity (Krisky et al., Gene Ther. 5, 1593-603 (1998); Samaniego et al., J. Virol. 72, 3307-20 (1998); Samaniego et al., J. Virol. 71, 4614-25 (1997)).

[0155] qRT-PCR was used to examine the expression of selected early and late viral genes ( Figure 11 D). At 12 h post-infection, JΔNI2 generally expressed the highest levels of these genes, and JΔNI5 the lowest, similar to the pattern observed for IE genes. These results demonstrate that replacing the native ICP0 and ICP27 promoters with early promoters is insufficient to silence the viral genome in the absence of ICP4, whereas deletion of both genes significantly reduced residual gene expression.

[0156] JΔNI reporter gene expression

[0157] To determine whether the activity of the exogenous human ubiquitin C (UbC) promoter (p) at the deleted ICP4 locus, introduced into the JΔNI vector along with the mCherry reporter gene, was also downregulated in JΔNI3- and JΔNI5-infected cells compared to JΔNI2-infected cells, mCherry expression was examined in infected HDFs. At 24 h post-infection with 5,000 gc / cell, mCherry fluorescence was readily detected in JΔNI2-infected cells but not in JΔNI3- or JΔNI5-infected cells ( Figure 12 A). Measurement of mCherry mRNA levels at 6 hpi by qRT-PCR revealed an approximately 100-fold reduction in transcriptional activity from the UbC promoter between JΔNI2- and JΔNI3-infected cells, and an additional approximately 5-fold reduction in JΔNI5-infected cells ( Figure 12 B); The KNTc control virus used for this experiment was replication competent and contained the UbCp-mCherry cassette in the intergenic region between UL3 and UL4. These data are consistent with the interpretation that residual ICP0 and ICP27 expression from JΔNI2 ( Figure 11 C) is sufficient to prevent complete transcriptional silencing of the viral genome, while deletion of both genes essentially eliminates all natural ( Figure 11 D) and exogenous ( Figure 12 A, 12B) Promoter activity. In contrast to HDFs, standard U2OS cells infected with JΔNI3 or JΔNI5 showed strong mCherry expression ( Figure 12 C), suggesting that the ICPO complement activity of U2OS cells is sufficient to activate these otherwise silenced genomes. To confirm that the viral ICPO protein has the same activity, JΔNI5-infected HDFs were repeatedly infected with ICP0+QOZHG virus (see Figure 11 B). Figure 12 As shown in Figure D, superinfection induced mCherry expression, while control superinfection did not. Taken together, these results indicate that ICP27 is not required to derepress the silenced JΔNI5 genome, at least in the presence of sufficient ICPO expression or complementation activity, and suggest that minimal amounts of ICPO expressed in JΔNI2-infected HDFs are sufficient to maintain limited transcriptional activity of the entire viral genome.

[0158] High-level transgene expression from the silenced JΔNI5 genome

[0159] Upon infection of neuronal cells, HSV enters a latent state in which the viral genome is transcriptionally silent except for the LAT locus. We investigated whether the LAT locus would similarly remain active in non-neuronal cells in the presence of an otherwise silent viral genome. To this end, an expression cassette consisting of the CAG enhancer / promoter and the EGFP gene (CAGp-GFP) was introduced into the LAT 2-kb intronic region of JΔNI5, generating a vector construct termed JΔNI7GFP ( Figure 13 As a control, the same CAGp-GFP cassette was introduced into the UL3-UL4 intergenic region of JΔNI5 to generate the vector JΔNI6GFP ( Figure 13 A); the UL3-UL4 intergenic region has been used for non-disruptive insertion of transgenic cassettes (Baines et al., J. Virol. 65, 938-44 (1991); Menotti et al., J. Virol. 76, 5463-71 (2002)) and is close to but outside the LAT locus. Elsewhere herein, "JΔNI6GFP" is referred to as "JΔNI6-CAGGFP" (see also Figure 1 A). Infectious virus was produced on U2OS-ICP4 / 27 cells and the gc:PFU ratio of fresh virus stocks was similar to that of JΔNI5 (Table 2). During amplification in U2OS-ICP4 / 27 cells, both JΔNI6-CAGGFP and JΔNI7GFP produced sufficient green and red fluorescence, demonstrating the integrity of their transgene expression cassettes. To examine the ability of these viruses to express the EGFP transgene in non-complementing cells, HDFs were infected with increasing gc / cell and EGFP fluorescence was recorded at 3 dpi ( Figure 13 B). JΔNI7GFP-infected cells showed sufficient, virus-dose-dependent EGFP expression, whereas JΔNI6-CAGGFP infection produced limited expression even at the highest dose. Despite the higher virus input used here compared to the earlier JΔNI5 infection, mCherry expression was minimal. These results were confirmed by qRT-PCR measurements of EGFP and mCherry mRNA levels at 3 and 5 dpi ( Figure 13 C) and is consistent with the suggestion that the JΔNI6-CAGGFP and JΔNI7GFP viral genomes, like the JΔNI5 genome, are silenced in infected HDFs while the LAT locus remains transcriptionally active, as EGFP mRNA levels in JΔNI7GFP-infected cells were at least as high at 5 dpi as at 3 dpi ( Figure 13 C), so we investigated whether expression could be detected at a later time when cells were fully contact inhibited. The results showed that expression persisted for at least 4 weeks in some cells ( Figure 13 D) Taken together, these observations indicate that the LAT locus is a preferred site for persistent transgene expression from HSV genomes that are free from functional loss of all IE gene expression without toxicity.

[0160] CTRL and LATP2 support transgene expression from the LAT locus

[0161] It has been shown that LAT expression during latency is controlled by latency-associated promoters (LAP) 1 and 2 (Goins et al., J. Virol. 68, 2239-52 (1994); Zwaagstra et al., Virology 182, 287-97 (1991)). LAP1 is located upstream of the transcription start site of the ~8.7-kb unstable primary LAT transcript that is processed into a stable 2-kb LAT intron, while LAP2 is located downstream of LAP1 in the first exon and extends into the 2-kb intron region. The region containing LAP2, which extends somewhat further into the intron, is called LATP2. In addition to acting as a promoter, the LAP2 / LATP2 region has been shown to function as a position-independent long-term expression / enhancer element for transgene expression in neurons (Berthomme et al., J. Virol. 74, 3613-22 (2000); Palmer et al., J. Virol. 74, 5604-18 (2000)). However, it has also been reported that during latency, the LAT locus is protected from global silencing by a region rich in CTCF binding sites, termed CTRLs, one located upstream of LAP1 (CTRL1) and the other in a 2-kb intron, just downstream of LATP2 (CTRL2) (Blooom et al., Biochim. Biophys. Acta 1799, 246-56 (2010)). Therefore, the possible role of LATP2 and CTRL elements in enabling the observation of EGFP expression in JΔNI7GFP-infected HDFs was explored. The three elements were deleted separately and in combination from the JΔNI7GFP genome ( FIG. 14A ) and reporter gene expression in infected HDFs was examined ( Figure 14B, 14C). Deletion of CTRL1 (ΔC1) or LATP2 (ΔLP2) resulted in a significant reduction in green fluorescence and EGFP mRNA levels, whereas deletion of CTRL2 (ΔC2) had only a small effect. Deletion of both CTRLs (ΔC12) had the same effect as deletion of CTRL1 alone, while deletion of all three elements (ΔC12LP2) further reduced expression to the levels observed in JΔNI6-CAGGFP-infected cells. mCherry fluorescence was undetectable in any infected cells ( Figure 14 B), suggesting that the different deletions do not cause derepression of other sites in the viral genome. These results indicate that both the CTRL1 and LATP2 regions play a significant role in protecting the linked transgene from transcriptional silencing in the context of a viral genome functionally lacking all IE genes.

[0162] LAT locus protection of transgene expression is independent of position

[0163] To determine whether sequences associated with the LAT locus are sufficient to protect the embedded transgene expression cassette from silencing in the absence of IE gene products, a restriction fragment corresponding to the LAT:CAGp-GFP region of JΔNI7GFP (including two CTRLs, LAP1 and LATP2) was inserted at one of two ectopic positions in JΔNI5 derivatives lacking the same LAT region to avoid recombination between the native and new sites. First, a Gateway (GW) recombination cassette was introduced into the intergenic region between UL45 and 46 (JΔNI9GW) or between UL50 and UL51 (JΔNI10GW) of the LAT-deleted JΔNI5 genome, and subsequently the LAT:CAGp-GFP fragment or CAGp-GFP without the LAT sequence was introduced by recombination with the GW cassette ( Figure 15 A). After virus production on U2OS-ICP4 / 27 cells (Table 2), the vectors were tested for reporter gene expression in infected HDFs. At 3 dpi, vectors lacking the LAT sequence surrounding the reporter cassette (JΔNI9GFP and JΔNI10GFP) showed low levels of EGFP fluorescence in infected cells ( Figure 15 B) and mRNA ( Figure 15 C), similar to the JΔNI6-CAGGFP control vector, indicating that the two intergenic insertion sites are transcriptionally repressed, similar to the intergenic region between UL3 and UL4 ( Figure 13 However, when the reporter cassette was flanked by LAT sequences on both sides (vectors JΔNI9LAT-GFP and JΔNI10LAT-GFP), EGFP expression increased to the levels observed in JΔNI7GFP-infected cells ( Figure 15B), indicating that the anti-silencing activity of the LAT-derived region functions in a position-independent manner. To confirm the dependence of this activity on LATP2 and either or both CTRLs, LATP2- and CTRL-deleted versions of the LAT:CAGp-GFP fragment were introduced into the JΔNI10GW genome by GW recombination; the deletions were identical to those in the LAT region of the earlier JΔNI7GFPΔC12LP2 vector ( Figure 14 A). This deletion reduced EGFP expression in infected HDFs, although not completely to the levels observed with the LAT-less JΔNI10GFP vector ( Figure 15 C, 15D; it is unclear why the ΔC12LP2 deletion appears to have a greater abundance here than in JΔNI7GFP (~50-fold, Figure 14 C) showed a smaller effect (-3.5-fold). However, these results clearly indicate that the portion of the LAT locus that includes the two CTRLs, LAP1 and LATP2, can protect the embedded transgene expression cassette from global silencing of the viral genome in the absence of IE gene expression in a position-independent manner, and suggest that at least CTRL1 and LATP2 play a role in this activity.

[0164] LAT locus elements protect transgene expression in other cell types

[0165] To evaluate the applicability of these findings beyond HDFs, EGFP and mCherry expression from the selected vectors was tested in other cell types. By qRT-PCR, higher EGFP mRNA levels were observed in JΔNI7GFP-infected human cells compared to JΔNI6-CAGGFP-infected human cells at 3 dpi ( Figure 16 B). The greatest difference was observed in BJ human foreskin fibroblasts (-35-fold), similar to that in HDFs (-30-fold, Figure 13 C), and in human hepatocytes (hHEP) (~40-fold). Human neonatal keratinocytes (hEK) showed the smallest difference (~4.5-fold), with intermediate values ​​observed in muscle-derived stem cells (hMDSC) (~10-fold) and preadipocytes (hPAD) (~7-fold). Comparison of the two vectors in fetal rat dorsal root ganglion (rDRG) neurons showed only a ~2.5-fold difference. Figure 16 A shows representative fluorescence images from independent experiments performed under the same infection conditions at 3 dpi. Figure 16The results were generally consistent with the qRT-PCR data, except for the different donors in B. Interestingly, while none of the human cells showed significant mCherry fluorescence, sufficient mCherry expression was observed in both JΔNI6-CAGGFP and JΔNI7GFP infected rat DRG cultures. It is unknown whether this result is unique to neural cells and could be a function of the promoter preceding the mCherry gene, the location of the expression cassette in the viral genome, and / or the rat origin of the cells. Maintaining hMDSCs for extended periods of time allowed monitoring of EGFP expression in JΔNI7GFP infected cells over time. Figure 16 As shown in C, EGFP was readily detectable in JΔNI7GFP-infected cells (but not in JΔNI6GFP-infected cells) for at least 4 weeks post-infection, similar to the observations made with JΔNI7GFP-infected HDFs ( Figure 13 D).

[0166] Finally, for some cells, it was determined whether CAGp-GFP activity in the UL50 / 51 intergenic region was enhanced by the flanking LAT sequence as in HDFs. Figure 16 The results in D show comparable enhancement in all three cell types, which, for unknown reasons, outweighs the difference between JΔNI6-CAGGFP and JΔNI7GFP in the same cells. Taken together, these results indicate that the position-independent anti-silencing activity of genetic elements in the LAT locus is not limited to HDFs but is also operational in a variety of non-neural human cell types.

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Example 9

[0177] This example shows the construction and properties of JΔNI8.

[0178] JΔNI8 was constructed by deleting UL41 from the above-mentioned JΔNI5 vector. Figure 17 JΔNI7GFP is essentially JΔNI5 with CAGp-EGFP-polyA inserted into the LAT locus between LATP2 and CTRL2. JΔNI8GFP is identical to JΔNI7GFP except for the deletion of UL41. See also Figure 20 .

[0179] The growth of JΔNI8 was evaluated relative to that of JΔNI5 in complementing cells, which were U2OS cells engineered to express ICP4 and ICP27 as described above. The complementing cells were infected with each vector at 1 viral genome copy (gc) / cell. Figure 18 As shown in Figure 2, JΔNI8 exhibited stronger growth than JΔNI5 as measured by plaque forming units (PFU), genome copies (gc), and expression of the mCherry transgene. In addition, it was observed that native HSV genes were expressed earlier during infection from JΔNI8 in complementing cells relative to JΔNI5 ( Figure 19 , represents the fold expression relative to each JDNI5 gene at 6 hpi which was arbitrarily assigned a value of 1).

[0180] As shown in Table 9-1, JΔNI8 and JΔNI8GFP produced more plaques per genome copy (gc) (on complementing cells) than JΔNI5 and JΔNI7GFP, respectively. This indicates that the ratio of functional to defective particles (a measure of mass) was higher in JΔNI8 and JΔNI8GFP preparations than in JΔNI5 and JΔNI7GFP preparations.

[0181] Table 9-1

[0182]

[0183] Furthermore, it was observed that infection with equal gc levels resulted in equal amounts of viral DNA in the nuclei of human dermal fibroblasts. Figure 10 For the discussed approach, this was achieved with much lower PFU for JΔNI8 compared to JΔNI5. Figure 21 ) These data suggest that infection with iso-gc should be used for studies comparing JΔNI8 to JΔNI5 (or JΔNI7GFP to JΔNI8GFP). MTT cell viability assay (see Figure 22) show that at 5 or 10 PFU / cell (MOI=5 or =10), JΔNI8 (upper-X-line in each figure) has measurably lower toxicity (approaching or exceeding the viability of uninfected control cells) than any other vector. The lower-X-line in each figure shows the results for JΔNI5. These data also show that vectors with an intact ICPO gene, even under the β promoter (JΔBBB3 (also known as JDBBB3 or JΔβββ3 or JDβββ3)) or in the absence of UL41 (QOZHG), are more toxic than JΔNI5 or JΔNI8. As expected, replicating virus (KOS) kills cells. Additional MTT experiments using HDFs show that although JΔNI8 shows some toxicity at higher MOIs, it is much less than that of JΔNI5 ( at a 3-fold lower MOI (equal gc / cell)). Figure 23 Other MTT data suggest that JΔNI8 is less toxic than JΔNI5 in some (HDFs, human neonatal keratinocytes, and human neural stem cells), but not all (Vero, human preadipocytes, and human hepatocytes) cell types (see Figure 24 ).

[0184] At 3 dpi, reporter gene expression from two transgenes (i.e., EGFP and mCherry) present in JΔNI7GFP and JΔNI8GFP, respectively, was compared in HDF cells. The results are shown in Figures 25 and 26. It was observed that GFP expression by JΔNI8GFP increased with the dose (from 5000 to 50,000 gc / cell, Figure 25 ) increased, but plateaued for JΔNI7GFP. Without wishing to be bound by theory, it is believed that the observed plateauing expression from JΔNI7GFP may be due to high-dose toxicity of the vector in HDF cells, suggesting that such toxicity is lower for JΔNI8GFP. In addition, significant mCherry expression was observed from HDF cells infected with JΔNI8GFP, but not from HDF cells infected with JΔNI7GFP. Similar experiments were performed using a constant dose of JΔNI7GFP or JΔNI8GFP (25,000 gc / cell), and the levels of EGFP or mCherry fluorescence were measured over time (2, 4, and 6 days post-infection). Figure 26 The reporter signal in JΔNI8GFP-infected cultures decreased between 4 and 6 dpi, likely due to dilution of the viral genome by intact cell division. The signal was more stable in JΔNI7GFP-infected cultures, likely due to reduced cell division.

[0185] Transgene expression (EGFP or mCherry) from other cells infected with JΔNI7GFP versus JΔNI8GFP was also investigated. Figure 27 As described in , JΔNISGFP expressed more GFP than JΔNI7GFP in human neonatal keratinocytes at two measured doses (12,500 gc / cell and 25,000 gc / cell).

[0186] In addition, transgene expression was determined from rat dorsal root ganglion (DRG) cells infected with JΔNI7GFP or JΔNI8GFP (at 6250 gc / cell). Figure 28 As described in [ 15 ], mCherry expression from JΔNI7GFP (inserted into the ICP4-deleted locus of both vectors) was enhanced in rat DRG neurons relative to GFP compared to non-neuronal cells and was relatively greater in JΔNI8GFP-infected cells than in JΔNI7GFP-infected cells. These results indicate that JΔNI8 is non-toxic and provides transgene expression from both the LAT and ICP4 loci in neurons.

[0187] Example 10

[0188] This example shows in vivo expression from the JΔNI7-GFP vector in a long-term experiment.

[0189] JDNI7-GFP (8.0x10 8 Genome copies / 2μl: 3.7x10 5 PFU) were stereotactically injected into the hippocampus of rats (AP: -1.8; ML: -1.7; P: +3.5). Animals were euthanized and perfused 6, 15 or 30 days after vehicle injection (dpi), and brain cryosections were imaged under a fluorescence microscope. Figure 29 Showing strong expression of EGFP and mCherry colocalized within the same cells in the hippocampus. Figure 29 Top, separate (left; 40x) or merged (center, right; 20x) images of EGFP and mCherry fluorescence at the indicated days post-infection (dpi); bottom, separate and merged images are depicted at 15 dpi (10x).

[0190] Example 11

[0191] This example involves the expression of the mCherry transgene from a JΔNI7-GFP vector in infected primary astrocytes.

[0192] Mouse astrocytes were infected (MOI = 5) and cultured. 25 days after infection, cells were fixed and exposed to an antibody specific for glial fibrillary acidic protein (GFAP), whose positive binding identifies astrocytes. Immunofluorescence imaging demonstrated that JΔNI7-GFP is non-toxic and provides persistent expression in astrocytes.

[0193] Example 12

[0194] This example demonstrates the generation of a cell line that complements the HSV vectors of the invention and also promotes the excision of a loxP-flanked BAC cassette during viral propagation.

[0195] The U2OS-ICP4 / 27 cell line discussed above was engineered to express Cre recombinase. The resulting cell line, U2OS-ICP4 / 27 / Cre, complements the function of all deficient HSV IE genes in vectors JDNI5, 7, 8, and their derivatives, except for the nonessential ICP47 gene, and removes the BAC cassette during viral growth.

[0196] U2OS-ICP4 / 27 / Cre cells were generated by infecting U2OS-ICP4 / 27 cells with a retroviral vector expressing Cre. For the construction of the retroviral vector, the plasmid pCX4Hyg (PNAS 2003, 100: 13567-13572; GenBank accession number AB086387) was modified by inserting the Gateway recombination cassette into the multiple cloning site to generate pCX4Hyg-GW. The complete NLS-Cre coding sequence from the plasmid pTurbo-Cre (GenBank accession number AF334827.1) was inserted between the attL1 and attL2 sites of pENTR1A (Invitrogen) and then transferred into pCX4Hyg-GW by LR Clonase-mediated Gateway recombination to generate pCX4Hyg-Cre. The plasmid and its construction are schematically shown in Figure 30 and 31 Retroviral particles were generated as described in [ 15 ] . Retroviral particles were generated as described by Makino et al. ( Exp Cell Res 2009, 315: 2727-2740 ). Briefly, pCX4Hyg-Cre was co-transfected with Gag-Pol and VSV-G expression plasmids into 293T cells, and the supernatant was collected 48 hours later, filtered through a 0.45 µm filter, and concentrated by centrifugation. Appropriate Gag-Pol and VSV-G plasmids are commercially available (e.g., pCMV-Gag-Pol (Cat. No. RV-111) from Cell Biolabs and pCMV-VSV-G from Addgene).

[0197] U2OS-ICP4 / 27 cells were infected with purified Cre retrovirus, and the cells were selected for resistance to puromycin, blasticidin, and hygromycin. Resistant clones were isolated, expanded, infected with JΔNI7-GFP at 0.001-1 PFU / cell, and stained for β-galactosidase activity 2 days after infection (dpi). It was found that clones showing few blue cells experienced a rapid cytopathic effect (100% CPE 4 days after infection at MOI = 0.01), while parental U2OS-ICP4 / 27 cells showed plaque-forming clusters of blue cells and approximately 25% CPE at 9dpi. These results indicate that Cre-mediated removal of the BAC element together with the LacZ expression cassette connected in JΔNI7-GFP promotes viral growth. Accurate removal of the BAC and LacZ sequences between the loxP sites of JΔNI7-GFP has been achieved by the selection of U2OS-ICP4 + / 27 + / Cre + PCR confirmation of viral DNA on individual JΔNI7-GFP plaques.

[0198] Thus, it has been observed that passaging of this U2OS-ICP4 / 27 / Cre cell line removes approximately 11 KB of BAC sequence, which allows room for transgene recovery and significantly accelerates viral growth.

[0199] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0200] The terms "a," "an," "the," "at least one," and similar referents, used in the context of describing the present invention (especially in the context of the following claims), are to be understood to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") is to be understood to mean one item (A or B) or a combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be understood as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise indicated. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary terminology (e.g., "such as"), are intended solely to better illustrate the invention and do not limit the scope of the invention unless otherwise required. No term in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0201] Preferred embodiments of the present invention are described herein, including the best mode of carrying out the present invention known to the inventors. After reading the foregoing description, changes to those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors expect that those skilled in the art will utilize these changes as appropriate, and the inventors intend that the present invention be practiced beyond what is specifically described herein. Therefore, as permitted by applicable law, the present invention includes all changes and equivalents of the subject matter described in the claims appended hereto. In addition, any combination of the above elements in all their possible variations is encompassed by the present invention, unless otherwise indicated herein or clearly contradicted by the context.

Claims

1. A herpes simplex virus vector comprising a polynucleotide encoding a transgene and the following genetic perturbations relative to wild-type herpes simplex virus: (a) Inactivating deletion of the gene encoding ICP0, (b) inactivating deletion of the gene encoding ICP4, (c) loss of joints, (d) The gene encoding ICP22 is under the control of the early promoter, (e) inactivating deletion of the gene encoding ICP27, and (f) Deletion of the promoter and start codon of the gene encoding ICP47, wherein ICP22 is expressed as an early gene, wherein the inactivating deletion in the gene encoding ICPO, the gene encoding ICP4, and the gene encoding ICP27 is a complete deletion of the coding sequence of each of said genes, wherein the herpes simplex virus vector comprises a latency-associated transcript gene region, wherein the latency-associated transcript gene region comprises a chromatin boundary element CTRL1 sequence, a chromatin boundary element CTRL2 sequence and a LATP2 or LAP2 enhancer element, and The polynucleotide encoding the transgene is inserted between the chromatin boundary element CTRL1 sequence and the chromatin boundary element CTRL2 sequence.

2. The herpes simplex virus vector of claim 1, wherein the transgene is transcribed in non-complementing cells, which are cultured human dermal fibroblasts.

3. The herpes simplex virus vector of claim 1, wherein the polynucleotide encoding the transgene is inserted between the chromatin boundary element CTRL2 sequence and the LATP2 enhancer element.

4. The herpes simplex virus vector of claim 1, further comprising an inactivating deletion in one or more non-essential genes.

5. The herpes simplex virus vector of claim 4, wherein the one or more non-essential genes comprises UL41.

6. The herpes simplex virus vector of claim 1, further comprising a gene encoding a mutant glycoprotein, wherein the mutant glycoprotein enhances the infectivity of the herpes simplex virus vector relative to a herpes simplex virus vector comprising a wild-type glycoprotein or directs the herpes simplex virus vector to enter cells through a non-canonical receptor.

7. The herpes simplex virus vector of claim 6, wherein the glycoprotein is selected from the group consisting of gB, gC, gD, gH and gK.

8. The herpes simplex virus vector of claim 1, wherein the transgene comprises a constitutive mammalian promoter.

9. The herpes simplex virus vector of claim 1, wherein the transgene comprises a cell- or tissue-specific promoter.

10. The herpes simplex virus vector of claim 1, wherein the polynucleotide encoding the transgene is polycistronic.

11. The herpes simplex virus vector of claim 1, wherein the polynucleotide encoding the transgene comprises one or more binding sites for microRNA.

12. The herpes simplex virus vector of claim 1, wherein the transgene is selected from the group consisting of Oct4, Klf4, Sox2, c-Myc, L-myc, dominant negative p53, Nanog, Glis1, Lin28, TFIID, GATA4, Nkx2.5, Tbx5, Mef2C, Myocd, Hand2, SRF, Mesp1, SMARCD3, SERCA2a, Pax3, MyoD, Lhx2, FoxG1, FoxP2, Isl1, Ctip2, Tbr1, Ebf1, Gsx2, Srebp2, Factor VIII, Factor IX, dystrophin, CFTR, GlyRα1, enkephalin, GAD isoforms, neurotrophins, Ascl1, Nurr1, Lmx1A, Brn2, Myt11, NeuroD1, FoxA2, ​​Hnf4a, Foxa1, Foxa2, Foxa3, miRNA or non-coding RNA.

13. The herpes simplex virus vector of claim 1, wherein the herpes simplex virus vector further comprises one or two consensus sequences for a recombinase.

14. The herpes simplex virus vector of claim 13, wherein the consensus sequence of the recombinase is inserted into an intergenic region.

15. A viral stock comprising the herpes simplex virus vector according to claim 1.

16. A pharmaceutical composition comprising the herpes simplex virus vector according to claim 1 and a pharmaceutically acceptable carrier.

17. A method for expressing a transgene in cells in vitro, the method comprising infecting non-complementing cells with the herpes simplex virus vector of claim 1 such that the transgene is expressed in the cells.

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