University 06350 Leydig 771537 1 HSV VECTORS CROSS-REFERENCE TO RELATED APPLICATION [0001] This patent application claims the benefit of U.S. Provisional Patent Application No.63/582,120, filed September 12, 2023, which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with Government support under Grant Number CA222804 awarded by the National Institutes of Health. The Government has certain rights in this invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 7,110 Byte Extensible Markup Language (XML) file named “771537_ST26.xml,” created September 4, 2024. BACKGROUND OF THE INVENTION [0004] Among many viral and non-viral genetic vector systems, Herpes Simplex Virus (HSV)-based vectors (e.g., replication defective (rd)HSV vectors) have been investigated for use as gene transfer vectors, including for possible therapeutic use in human patients. HSV is a complex, non-integrating DNA virus capable of infecting a very wide range of human and animal cells. In the past, it has proven to be challenging to provide an rdHSV vector that is not capable of expressing native genes which cause human infection, and at the same time be effective at expression of a desired transgene over a period of time long enough to provide therapeutic effects. [0005] Accordingly, there remains a need for a safe and effective HSV vector capable of long-term expression of a transgene in any tissue or cell, in vitro or in vivo.
University 06350 Leydig 771537 2 BRIEF SUMMARY OF THE INVENTION [0006] An aspect of the invention provides a recombinant herpes simplex virus (HSV) vector comprising (a) an insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in an intergenic region, a LAT locus, or an ICP4 locus of the vector, (b) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP0 or (b) comprises one or more sequences that encode one or more ICP0 genes, with each sequence that encodes the one or more ICP0 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP0 genes, (c) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP4 or (b) comprises one or more sequences that encode one or more ICP4 genes, with each sequence that encodes the one or more ICP4 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP4 genes, (d) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP27 or (b) comprises one or more sequences that encode one or more ICP27 genes, with each sequence that encodes the one or more ICP27 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP27 genes, (e) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP47 or (b) comprises one or more sequences that encode one or more ICP47 genes, with each sequence that encodes the one or more ICP47 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP47 genes, (f) wherein the vector either (a) does not comprise one or more sequences that encode internal repeat (joint) region genes or (b) comprises one or more sequences that encode one or more internal repeat (joint) region genes, with each sequence that encodes the one or more internal repeat (joint) region genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more internal repeat (joint) region genes, and (g) wherein the vector either (a) does not comprise one or more sequences that encode gene virion host shut-off (UL41) or (b) comprises one or more sequences that encode one or more UL41 genes, with each sequence that encodes the one or more UL41 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more UL41 genes.
University 06350 Leydig 771537 3 [0007] Another aspect of the invention a pharmaceutical composition comprising the vector of an aspect of the invention, and a pharmaceutically acceptable carrier. [0008] Another aspect of the invention provides a method for administering one or more transgenes into a cell in a subject comprising providing the vector of an aspect of the invention, or the pharmaceutical composition of an aspect of the invention, to the subject. BRIEF DESCRIPTION OF THE DRAWINGS [0009] Figure 1A shows a schematic of a vector of an aspect of the invention. The JΔNI8L-ZsG/fLuc vector contains a transgene cassette expressing ZsGreen (ZsG) and firefly luciferase (fLuc), ZsG/fLuc, under the control of the CAG promoter in the LAT locus. This transgene cassette is surrounded by the viral endogenous insulator sequences (CTRL2, CTRL1, triangles) and the enhancer-like latency active promoter 2 (LATP2). JΔNI8L- ZsG/fLuc contains a second transgene cassette in the ICP4 locus with the Ubiquitin (UbC) driving the expression of the mCherry reporter gene. [0010] Figure 1B shows a schematic of a vector of an aspect of the invention. In the JΔNI84-ZsG/fLuc vector, the ZsG/fLuc cassette is located in the ICP4 locus. In this instance, the HSV endogenous insulator elements, CTRS3 (triangle) is located upstream of the ICP4 locus while the downstream endogenous insulator sequence (CTRS1/2, triangle) has been deleted. [0011] Figure 2A shows a set of images showing the level of native expression of the ZsG transgene from the LAT and ICP4 loci upon infection with JΔNI8L-ZsG/fLuc and JΔNI84-ZsG/fLuc vectors over a course of 21 days post-infection (dpi) of SH-SY5Y cells, a human neuroblastoma cell, with multiplicity of infection (MOI) of 5,000 gc/cell of vectors of an aspect of the invention. [0012] Figure 2B shows a graph showing the levels of luciferase (fLuc expression) of the cells of Figure 2A. fLuc expression was monitored for 21 dpi. Data are means and standard errors of the mean (SEM) of triplicate infections. Statistical differences were determined by two tailed non-parametric Mann-Whitney test (* p < 0.05; **** p < 0.0001). [0013] Figure 2C shows a graph showing the percent of survival of the cells of Figure 2A. ALAMARBLUE
TM cell viability assay was performed at the reported time points. Data
University 06350 Leydig 771537 4 are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0014] Figure 3A shows three representative images of mice injected with JΔNI84- ZsG/fLuc and JΔNI8L-ZsG/fLuc vectors of an aspect of the invention at MOI of 8*10
8 gc/mouse. Luciferase activity is shown at 3 dpi (top panel) and 120 dpi (lower panel). The images show the in vivo expression of fLuc upon stereotactic delivery into the mice dorsal hippocampus. The gray scale indicate activity levels. [0015] Figure 3B shows a graph showing the level of bioluminescent (BLI) signal expressed over time in the mice of Figure 3A following injection with JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc vectors of an aspect of the invention, or PBS. Data are an average of 6 mice per group and are expressed in photons per seconds (p/s). Statistical differences were determined by two tailed non-parametric Mann-Whitney test (* p < 0.05). [0016] Figure 4 shows ZsG distribution in the mouse hippocampus upon intraparenchymal (IP) delivery of JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc. Representative images (5 sections/animals, 5 animals/group) of coronal sections from animals injected with JΔNI84-ZsG/fLuc (top) or JΔNI8L-ZsG/fLuc (bottom) sacrificed at 5 months post-infection (mpi). Column A shows nuclei stained with DAPI; column B shows nuclei of neurons stained with NEUROTRACE
TM; column C shows ZsG positive cells; column D shows nuclei stained with DAPI, NEUROTRACE
TM and ZsG triple-fluorescence; column E shows higher magnifications of the framed white box areas shown in column D. [0017] Figure 5 shows ZsG and mCherry distribution in consecutive sections of the mouse hippocampus at 5 mpi. Representative images (5 sections/animal, 3 animals/group) taken from coronal sections prepared from animals injected with JΔNI8L-ZsG/fLuc and sacrificed at 5 mpi. Middle panels show low magnification images of the whole hippocampus; upper panels show higher magnification of the dentate gyrus, framed in white in the middle columns A and D; lower panels show higher magnifications of the CA3 areas, framed in white in the middle panel columns A and D. Columns A and D show nuclei stained with DAPI; column B shows nuclei stained with mCherry signal; column C shows nuclei stained with DAPI and mCherry double-fluorescence; column E shows nuclei stained with ZsG signal; column F shows nuclei stained with DAPI and ZsG double-fluorescence. Gray * in the lower panel of column E indicate ZsG positive cells that did not co-localize with mCherry-positive cells.
University 06350 Leydig 771537 5 [0018] Figure 6A shows a set of images transgene expression in a mouse myoblast cell line, C2C12. The C2C12 cells were infected upon differentiation at a MOI of 5,000 gc/cell with JΔNI84-ZsG/fLuc (top panel) or JΔNI8L-ZsG/fLuc (lower panel) vectors of an aspect of the invention. The native expression of the ZsG transgene from the ICP4 and LAT loci was monitored until 12 dpi. [0019] Figure 6B shows a graph showing the levels of luciferase (fLuc expression) of the cells of Figure 6A. Luciferase activities were monitored for 10 dpi. Data are means and SEM of triplicate infections. Statistical differences were determined by two tailed non- parametric Mann-Whitney test (* p < 0.05; **p < 0.001 **** p < 0.0001). [0020] Figure 6C is a graph showing the percent of survival of the cells of Figure 6A. ALAMARBLUE
TM cell viability assay was used at the indicated time points. Data are shown as percent survival compared to mock infected controls. Data are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0021] Figure 7A shows in vivo expression of fLuc upon direct injection (DI) of JΔNI84- ZsG/fLuc (left panels) or JΔNI8L-ZsG/fLuc (right panels) into the mice hind limb muscles. Luciferase activity was measured in 5 representative mice per group at 1 dpi (upper panel) and 365 dpi (lower panel). [0022] Figure 7B shows a graph showing the level of bioluminescent (BLI) signal expressed over time in mice of Figure 7A following injection with JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc vectors of an aspect of the invention. Data are an average of 8 mice per group and are expressed in photons per seconds (p/s). Background was calculated on bioluminescence signal emitted by back fur. Data are means and SEM per group. Statistical differences were determined by two tailed non-parametric Mann-Whitney test (* p < 0.05). [0023] Figure 8A shows hematoxylin and eosin–stained full cross section of the mouse hind limb. Black boxes indicate the areas of the muscle where ZsG positive signal is localized. [0024] Figure 8B shows ZsG distribution in representative cross section of the hind limb muscles (5 sections/animals, 5 animals/group) upon DI of JΔNI8L-ZsG/fLuc or JΔNI84- ZsG/fLuc at 365 dpi. Upper panels show low magnification images (4X); lower panels show higher magnifications of the areas framed in white in the upper panels (10X); (i) Nuclei, stained with DAPI; (ii) dystrophin staining; (iii) ZsG positive fibers; (iv) DAPI, dystrophin and ZsG triple-fluorescence.
University 06350 Leydig 771537 6 [0025] Figure 9A shows a schematic of an aspect of the invention. Specifically, this vector shows a control transgene cassette containing the ZsG and fLuc reporter genes under the control of the CAG promoter and was used to generate the control vectors JΔNI84- ZsG/fLuc and JΔNI8L-ZsG/fLuc by Gateway (GW) reaction. [0026] Figure 9B shows a schematic of an aspect of the invention. The expression cassette described in Figure 9A was flanked by the mouse tRNA for glutamine and glycine sequences (SEQ ID NO: 1, 2tRNA, 492 bp) and was separated by AT-rich sequences and oriented in an opposite direction upstream and downstream the expression cassette (2tR insulator design), and was used to generate JΔNI84-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR by GW reaction. [0027] Figure 9C shows a schematic of an aspect of the invention. The expression cassette described in Figure 9B with the matrix attachment region sequence (SEQ ID NO: 4, SMAR, 2 kb), derived from the human β-interferon gene cluster located between the fLuc stop codon and the BGH polyA (2tR-SMAR insulator design), was used to generate JΔNI84- ZsG/fLuc-2tR-SMAR and JΔNI8L-ZsG/fLuc-2tR-SMAR by GW reaction. [0028] Figure 10A shows a graph showing cells from U2OS 4/27 cell line used for viral propagation that were infected with vectors described in Figure 9A, 9B and 9C at MOI of 0.5 gc/cell and the supernatant were harvested from triplicate wells (n=3) at the indicated time points and titered by qPCR for U
L5 gene. [0029] Figure 10B shows a graph showing the level of luciferase expression in cells from a U2OS 4/27 cell line at the indicated time points. Data are presented as average and SEM of 3 biological replicates. [0030] Figure 11A shows a set of images showing in vitro analysis of JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors of an aspect of the invention, following entry and replication of the vectors in the cells. A U2OS 4/27 cell line was used for viral propagation. The cells were infected at MOI of 0.5 gc/cell and ZsG native expression was evaluated at several time points. [0031] Figure 11B shows a set of images showing in vitro analysis of JΔNI8L-ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR vectors entry and replication. A U2OS 4/27 line was used for viral propagation. The cells were infected at MOI of 0.5 gc/cell and ZsG native expression was evaluated at several time points.
University 06350 Leydig 771537 7 [0032] Figure 12A shows the effect of the 2tR and 2tR-SMAR insulator design on transgene expression upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84- ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc- 2tR-SMAR in infected human SH-SY5Y (5000 gc/cell) neuronal cell culture. Native expression of ZsG from the ICP4 or LAT locus in the presence or absence of cellular insulator elements monitored over a time course of 21 dpi. [0033] Figure 12B shows a graph showing the levels of luciferase (fLuc expression) of the cells of Figure 12A monitored for 21 dpi. Data are means and SEM of triplicate infections. Statistical differences were determined by two tailed non-parametric Mann- Whitney test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). [0034] Figure 12C is a graph showing the percent of survival of the cells of Figure 12A. ALAMARBLUE
TM cell viability assay was used at the indicated time points. Data are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0035] Figure 13A shows the effect of the 2tR and 2tR-SMAR cellular insulators designs located in the ICP4 locus on transgene expression in vivo. Luciferase activity was measured in 3 representative mice per group at 3 dpi (upper panel) and 168 dpi (lower panel). [0036] Figure 13B shows a graph showing the level of bioluminescent (BLI) signal expressed up to 6 mpi in mice of Figure 13A following injection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR. Dark gray, luciferase activity from the ICP4 locus flanked only by the viral insulator (JΔNI84-ZsG/fLuc; n = 6); light gray, luciferase activity from the ICP4 locus flanked by the 2tR insulator design (JΔNI84- ZsG/fLuc-2tR; n = 6); gray, luciferase activity from the ICP4 locus flanked by 2tR-SMAR insulator design (JΔNI84-ZsG/fLuc-2tR; n = 6); black circles, background calculated on BLI signal emitted by back fur. Data are means and SEM per group. Statistical differences were determined by ANOVA (* p < 0.05; ** p< 0.01). [0037] Figure 14 shows localization of ZsG expression from the ICP4 locus with or without the 2tR or 2tR-SMAR insulator designs in the mouse hippocampus. Representative images (5 sections/animals, 5 animals/group) of coronal sections from animals injected with JΔNI84-ZsG/fLuc (top panel); JΔNI84-ZsG/fLuc-2tR (middle panel) or JΔNI84-ZsG/fLuc- 2tR-SMAR (lower panel) sacrificed at 6 mpi. For each treatment group, the upper panels show low magnification images of the whole hippocampus; lower panels show higher
University 06350 Leydig 771537 8 magnifications of the CA3 areas, framed in white in the upper panels of column A. Column A shows nuclei stained with DAPI; column B shows nuclei of neurons stained with NEUROTRACE
TM; column C shows ZsG positive cells; column D shows DAPI, NEUROTRACE
TM and ZsG triple-fluorescence positive cells. [0038] Figure 15 shows the distribution of cells expressing ZsG from the ICP4 locus with or without the 2tR or 2tR-SMAR insulator designs in the mouse hippocampus. Top, five coronal sections across the hippocampus are shown, including their antero-posterior coordinates relative to bregma (Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego (1998)). Bottom left, representative images (5 sections/animals, 5 animals/group) of coronal sections from animals injected with JΔNI84- ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR or JΔNI84-ZsG/fLuc-2tR-SMAR sacrificed at 2 wpi. Bottom right, representative images (5 sections/animals, 5 animals/group) of coronal sections from animals injected with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR or JΔNI84-ZsG/fLuc- 2tR-SMAR sacrificed at 6 mpi. ZsG native expression is shown and nuclei stained with DAPI. [0039] Figure 16 shows a graph showing the level of bioluminescent (BLI) signal expressed over 11 mpi in 3 mice injected with JΔNI84-ZsG/fLuc-2tR-SMAR. [0040] Figure 17A shows a set of images showing the in vitro effect of the 2tR and 2tR- SMAR insulator design on transgene expression upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L- ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR in C2C12 (5000 gc/cell) mouse differentiated myoblast. Cells from a C2C12 mouse myoblast cell line were infected 7 days post differentiation at MOI of 5,000 gc/cell. ZsG native expression from the ICP4 or LAT loci in the presence or absence of cellular insulator elements was monitored over a time course of 10 dpi. [0041] Figure 17B shows a graph showing the level of luciferase expression in C2C12 cells following infection with JΔNI8 vectors described in Figures 9A, 9B, and 9C of an aspect of the invention. Cells from a C2C12 mouse myoblast cell line were infected 7 days post differentiation at MOI of 5,000 gc/cell and luciferase activities were monitored for 10 dpi. Data are means and SEM of triplicate infections. Statistical differences were determined by two tailed non-parametric Mann-Whitney test (** p < 0.01).
University 06350 Leydig 771537 9 Figure 17C shows a graph showing the survival level of C2C12 cells upon infection with JΔNI8 vectors described in Figures 9A, 9B, and 9C with an equivalent genome copy input. Cells from a C2C12 mouse myoblast cell line were infected 7 days post differentiation at MOI of 5,000 gc/cell and cell survival was evaluated by ALAMARBLUE
TM cell viability assay. Data are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0042] Figure 18 shows a set of images showing ZsG native expression co-localized with dystrophin expression in differentiate C2C12 cells. Cells from a C2C12 cell line were stained with dystrophin upon infection with JΔNI84-ZsG/fLuc, JΔNI8L-ZsG/fLuc, JΔNI84- ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc- 2tR-SMAR vectors of an aspect of the invention. ZsG native expression is shown, and nuclei were stained with DAPI. [0043] Figure 19A shows the effect of the 2tR and 2tR-SMAR cellular insulator designs located in the LAT locus on the in vivo expression of fLuc upon DI into the mice hind limb muscles. Black, luciferase activity from the LAT locus flanked only by the viral insulators (JΔNI8L-ZsG/fLuc; n = 6); light gray, luciferase activity from the LAT locus flanked by the 2tR design (JΔNI8L-ZsG/fLuc-2tR; n = 6); gray, luciferase activity from the LAT locus flanked by the 2tR-SMAR insulator design (JΔNI8L-ZsG/fLuc-2tR-SMAR; n = 6). Black circles, background calculated on bioluminescence signal emitted by back fur. Data are means and SEM per group. Statistical differences were determined by ANOVA (* p < 0.05). [0044] Figure 19B shows the effect of the 2tR and 2tR-SMAR cellular insulator designs located in the ICP4 locus on the in vivo expression of fLuc upon DI into the mice hind limb muscles. Gray, luciferase activity from the viral insulators present in the ICP4 locus (JΔNI84- ZsG/fLuc; n = 6); light gray, luciferase activity from the 2tR design in the ICP4 locus (JΔNI84-ZsG/fLuc-2tR; n = 6); black, luciferase activity from the 2tR-SMAR design in the ICP4 locus (JΔNI84-ZsG/fLuc-2tR-SMAR; n = 6). Black circles, background calculated on bioluminescence signal emitted by back fur. Data are means and SEM per group. Statistical differences were determined by ANOVA (* p < 0.05). [0045] Figure 20 shows a schematic representation of a dual-reporter, IE gene deficient vector JΔNI7GFP-GW previously described in (Miyagawa, et al., PNAS, 112(13): E1632- E1641 (2015)). The fLuc gene was introduced under the control of the CAG promoter, with or without, 2tR-SMAR into the UL50-UL51 intergenic region via a GW reaction. The
University 06350 Leydig 771537 10 resulting viruses, JΔNI7GFP-fLuc and fLuc-2tR-SMAR were tested in the differentiated SH-SY5Y and C2C12 cells. [0046] Figure 21A shows the luciferase activities in SH-SY5Y cells upon infection (5000 gc/cell) with JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR monitored for 21 dpi. Data are means and SEM of triplicate infections. Statistical differences were determined by two tailed non-parametric Mann-Whitney test (*** p < 0.001; **** p < 0.0001). [0047] Figure 21B is a graph showing the percent of survival of the cells of Figure 21A. ALAMARBLUE
TM cell viability assay was used at the indicated time points. Data are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0048] Figure 22A shows luciferase activities in C2C12 cells upon infection (5000 gc/cell) with JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR monitored for 10 dpi. Data are means and SEM of triplicate infections. Statistical differences were determined by two tailed non-parametric Mann-Whitney test (** p < 0.01; **** p < 0.0001). [0049] Figure 22B is a graph showing the percent of survival of the cells of Figure 22A. ALAMARBLUE
TM cell viability assay was used at the indicated time points. Data are shown as percent survival compared to mock infected controls. Data are means and SEM of triplicate infections. [0050] Figure 23 shows dystrophin positive fibers in the hind limb muscles of DBA wild- type mice injected with PBS and D2.mdx mice injected with either JΔNI7-mDMD (Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)) or PBS at 2 wpi. Representative cross section of hind limb muscles (8 sections/animals, 3 animals/group). Groups and treatments are reported on the left. Upper panels show low magnification images (4X); lower panels show higher magnifications of the areas framed in white in the upper panels of column A; Column A shows nuclei stained with DAPI; column B shows dystrophin positive fibers; and column C shows DAPI and dystrophin double-fluorescence. [0051] Figure 24 shows dystrophin distribution in the hind limb muscle of D2.mdx mice upon DI of JΔNI7-mDMD. Representative cross section of hind limb muscle (8 sections/animals, 3 animals) injected with JΔNI7-mDMD and sacrificed at 2 wpi. Middle panels show low magnification images (4X); upper panels show higher magnifications of the muscle area where abundant dystrophin transduced fiber are observed (framed in white in the middle panel of column A); lower panels show higher magnifications of the muscle area
University 06350 Leydig 771537 11 where a clear distinction between dystropin transduced and untransduced fibers are observed (framed in white in the middle panel of column A. Column A shows nuclei stained with DAPI; column B shows dystrophin positive fibers; column C shows DAPI and dystrophin double-fluorescence. White * in the bottom panel of column A is indicating the limit between dystrophin transduced and untransduced fibers. DETAILED DESCRIPTION OF THE INVENTION [0052] An aspect of the invention is directed to safe and effective HSV vectors that provide robust and prolonged transgene expression. The vectors of an aspect of the invention comprise an insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in an intergenic region, a LAT locus, or an ICP4 locus of the vector. A sequence comprising at least 95% identity to SEQ ID NOs: 1-4, used alone or in combination with another sequence comprising at least 95% identity to SEQ ID NOs: 1-4, allows the vectors to provide expression of the desired transgenes. The insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, alone or in combination with another insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, unexpectedly protects the transgene from heterochromatin silencing. [0053] In an aspect of the invention, the HSV vector is a replication-defective (rd)HSV. rdHSV vectors are HSV vectors which include at least one mutation or deletion in at least one gene essential for viral replication (e.g., origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), a helicase/primase complex (UL5/UL8/UL52) genes), and the immediate early (IE) genes ICP4 and ICP27). The vectors of an aspect of the invention comprise one or more deletions or one or more mutations that make the vectors unable to replicate outside of an engineered cell line, which complements in trans the one or more deleted genes or the one or more mutated genes. Specifically, the JΔNI vectors of an aspect of the invention comprise a deletion for the joint region (ΔJOINT). The deleted joint region includes the sequences comprising the ICP47 promoter, translation initiation codon, and the ICP4 and ICP0 immediate early (IE) genes. The second copies of (a) the ICP0 gene (located in the long terminal repeat (TR
L)) and (b) the ICP4 gene (located in the short terminal repeat (TRS)) have been deleted. In the JΔNI vectors of an aspect of the invention, the ICP27 gene has also been deleted. In addition, the ICP22 IE
University 06350 Leydig 771537 12 is converted to early-expression kinetics by deletion of the promoter TAATGARAT in the JΔNI vectors of an aspect of the invention. [0054] The vectors of an aspect of the invention also have the following unexpected features. [0055] Previously, cells infected with HSV vectors (e.g., rdHSV vectors) tended to show level or progressively reduced expression of transgenes over time. However, the vectors of an aspect of the invention actually show increased expression of the transgenes over time. Further, the vectors of an aspect of the invention also provide for transgene expression for an extended period of time. [0056] The insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 can provide enhanced expression of the transgenes when placed in different positions within the vectors of an aspect of the invention. For example, the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 can be placed within a LAT locus, an ICP4 locus, or within an intergenic region of the vector. [0057] The vectors of an aspect of the invention also can provide enhanced transgene expression when in many different cell types. For example, the vectors of an aspect of the invention have been found to be effective in muscle cells (prior to and after myotube formation) and neuron cells. [0058] In addition, the vectors of an aspect of the invention are safe to use. The vectors of an aspect of the invention did not show any cell toxicity and did not produce a HSV infection. [0059] Due to the protection from heterochromatin silencing, the vector does not require HSV protein ICP0 to be expressed in order to express the transgene. Further, the vectors of an aspect of the invention do not express any immediate early HSV genes such as ICP4, ICP27, and ICP47. [0060] The vectors of an aspect of the invention also do not express internal repeat (joint) region genes which comprise IRS and IRL. The absence of the expression of the joint region can contribute to the stability of the vector. Deleting the joint region also allows for the vectors of an aspect of the invention to accommodate large transgenes (at least 15 kb) and still be packaged correctly into mature virions. Further, deletion of the joint region eliminates one copy each of the IE genes ICP0 and ICP4 such that the remaining copies of these genes
University 06350 Leydig 771537 13 can be more easily manipulated. Deletion of the joint region also deletes the promoter for the ICP22 and ICP47 immediate early gene. [0061] The vectors of an aspect of the invention also do not express gene virion host shut-off (vhs) UL41. UL41 is an RNAse that degrades many host and viral mRNAs, causes rapid shutoff of host cell protein synthesis, and enters cells as a virion tegument component. Vectors [0062] An aspect of the invention provides a recombinant HSV vector (e.g., rdHSV vector) comprising (a) an insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in an intergenic region, a LAT locus, or an ICP4 locus of the vector, (b) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP0 or (b) comprises one or more sequences that encode one or more ICP0 genes, with each sequence that encodes the one or more ICP0 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP0 genes, (c) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP4 or (b) comprises one or more sequences that encode one or more ICP4 genes, with each sequence that encodes the one or more ICP4 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP4 genes, (d) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP27 or (b) comprises one or more sequences that encode one or more ICP27 genes, with each sequence that encodes the one or more ICP27 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP27 genes, (e) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP47 or (b) comprises one or more sequences that encode one or more ICP47 genes, with each sequence that encodes the one or more ICP47 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP47 genes, (f) wherein the vector either (a) does not comprise one or more sequences that encode internal repeat (joint) region genes or (b) comprises one or more sequences that encode one or more internal repeat (joint) region genes, with each sequence that encodes the one or more internal repeat (joint) region genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more internal repeat
University 06350 Leydig 771537 14 (joint) region genes, and (g) wherein the vector either (a) does not comprise one or more sequences that encode gene virion host shut-off (UL41) or (b) comprises one or more sequences that encode one or more UL41 genes, with each sequence that encodes the one or more UL41 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more UL41 genes. [0063] Any suitable HSV strain can be used as a vector of an aspect of the invention. The genome sequences of many HSV strains are known to persons of ordinary skill (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). Accordingly, manipulation of the sequence of HSV genes and loci is within the level of ordinary skill. It should also be noted that these published sequences are merely exemplary and that other strains or variants of HSV can be employed as a source genome in engineering the inventive vector. In an aspect of the invention, the HSV vector is a rdHSV vector. [0064] In an aspect of the invention, the vector comprises an insulator sequence comprising at least about 95% (i.e., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%) identity to SEQ ID NO: 1. SEQ ID NO: 1 are two murine transfer RNA sequence separated by a region rich in A and T nucleotide. If present in the vector, SEQ ID NO: 1 (or a sequence comprising at least 95% identity to SEQ ID NO: 1) may be present in the vector one or more than one times (e.g., 1, 2, 3, 4, or 5 more or times). [0065] In an aspect of the invention, the vector comprises an insulator sequence comprising at least about 95% (i.e., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%) identity to SEQ ID NO: 2. SEQ ID NO: 2 is a murine transfer RNA sequence for glycine. If present in the vector, SEQ ID NO: 2 (or a sequence comprising at least 95% identity to SEQ ID NO: 2) may be present in the vector one or more than one times (e.g., 1, 2, 3, 4, or 5 more or times). SEQ ID NO: 2 is present within SEQ ID NO: 1. [0066] In an aspect of the invention, the vector comprises an insulator sequence comprising at least about 95% (i.e., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%) identity to SEQ ID NO: 3. SEQ
University 06350 Leydig 771537 15 ID NO: 3 is a murine transfer RNA for glutamine. If present in the vector, SEQ ID NO: 3 (or a sequence comprising at least 95% identity to SEQ ID NO: 3) may be present in the vector one or more than one times (e.g., 1, 2, 3, 4, or 5 more or times). SEQ ID NO: 3 is present within SEQ ID NO: 1. [0067] In an aspect of the invention, the vector comprises an insulator sequence comprising at least about 95% (i.e., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%) identity to SEQ ID NO: 4. SEQ ID NO: 4 is a scaffold/matrix attachment region (SMAR) and is a sequence of DNA in eukaryotic chromosomes where nuclear matrix attaches. If present in the vector, SEQ ID NO: 4 (or a sequence comprising at least 95% identity to SEQ ID NO: 4) may be present in the vector one or more than one times (e.g., 1, 2, 3, 4, or 5 more or times). [0068] The vector of an aspect of the invention may comprise any combination of insulator sequences comprising at least 95% identity to SEQ ID NOs: 1-4 (i.e., at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NOs: 1-4). For example, the vector of an aspect of the invention may comprise two or more sequences, e.g., (i) a sequence comprising at least 95% identity to SEQ ID NO: 1 and a sequence comprising at least 95% identity to SEQ ID NO: 2, (ii) a sequence comprising at least 95% identity to SEQ ID NO: 1 and a sequence comprising at least 95% identity to SEQ ID NO: 3, (iii) a sequence comprising at least 95% identity to SEQ ID NO: 1 and a sequence comprising at least 95% identity to SEQ ID NO: 4, (iv) a sequence comprising at least 95% identity to SEQ ID NO: 2 and a sequence comprising at least 95% identity to SEQ ID NO: 3, (v) a sequence comprising at least 95% identity to SEQ ID NO: 2 and a sequence comprising at least 95% identity to SEQ ID NO: 4, or (vi) a sequence comprising at least 95% identity to SEQ ID NO: 3 and a sequence comprising at least 95% identity to SEQ ID NO: 4. The vector of an aspect of the invention may comprise three or more sequences, e.g., (i) a sequence comprising at least 95% identity to SEQ ID NO: 1, a sequence comprising at least 95% identity to SEQ ID NO: 2, and a sequence comprising at least 95% identity to SEQ ID NO: 3; (ii) a sequence comprising at least 95% identity to SEQ ID NO: 1, a sequence comprising at least 95% identity to SEQ ID NO: 2, and a sequence comprising at least 95% identity to SEQ ID NO: 4; (iii) a sequence comprising at least 95% identity to SEQ ID NO: 1, a sequence comprising at least 95% identity to SEQ ID NO: 3, and a sequence comprising at least 95% identity to SEQ ID NO: 4; or (iv) a sequence comprising
University 06350 Leydig 771537 16 at least 95% identity to SEQ ID NO: 2, a comprising at least 95% identity to SEQ ID NO: 3, and a sequence comprising at least 95% identity to SEQ ID NO: 4. The vector of an aspect of the invention may comprise four sequences, i.e., sequences comprising at least 95% identity to SEQ ID NOs: 1-4 (a sequence comprising at least 95% identity to SEQ ID NO: 1, a sequence comprising at least 95% identity to SEQ ID NO: 2, a sequence comprising at least 95% identity to SEQ ID NO: 3, and a sequence comprising at least 95% identity to SEQ ID NO: 4). [0069] In an aspect of the invention, SEQ ID NOs: 1-4 (or a sequence comprising at least 95% identity to any of SEQ ID NOs: 1-4), if present in the vector, are independently positioned within an intergenic region, a LAT locus, or an ICP4 locus of the vector. For example, in Figure 9B (left) a vector is shown with an insulator sequence comprising SEQ ID NO: 1 in a LAT locus. Also, Figure 9B (right) shows a vector with an insulator sequence comprising SEQ ID NO: 1 in an ICP4 locus. Further, an insulator sequence comprising SEQ ID NO: 4 is shown in a LAT locus and a ICP4 locus of vectors in Figure 9C. It is known by one skilled in the art how to insert a sequence comprising each of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) into an intergenic region, a LAT locus, or an ICP4 locus of the vector. [0070] The vectors of an aspect of the invention do not express HSV genes ICP0, ICP4, ICP27, ICP22, UL47, and internal repeat (joint) region genes. There are several ways of modifying HSV so that these genes are not expressed. The vector (1) may not comprise one or more sequences that encode these genes, (2) may comprise one or more sequences that encode one or more of the genes, however, each sequence that encodes the one or more genes comprises an inactivating mutation within the sequence, and/or (3) may comprise an inactivating mutation within the promoter region that controls expression of the one or more genes. One or more of these HSV genes (i.e., ICP0, ICP4, ICP27, ICP22, UL47, and internal repeat (joint) region genes) can be engineered to be expressed as an early or late gene. For example, a vector of an aspect of the invention can be modified to retain the coding sequence of one of more of these genes, but the gene’s promoter can be changed to a different promoter thereby making the gene expressed as an early (beta) or late (gamma), but not immediate early (alpha) gene. For example, such gene can be placed under the control of a promoter responsive to ICP4. A suitable promoter for expressing such gene with early (beta) kinetics is the HSV tk promoter. The ICP22 promoter may be converted to early kinetics by
University 06350 Leydig 771537 17 truncation, i.e. deletion of regulatory The entire ICP47 promoter and initiation codon can be deleted. Alternatively, in an aspect of the invention, the ICP47 gene can be expressed as an immediate early gene to protect infected cells against immune recognition (Hill, et al., Nature, 375(6530): 411–415 (1995); Goldsmith, et al., J. Exp. Med., 187(3): 341–348 (1998)). It is known by one skilled in the art how to use routine methods to delete all or part of each of these genes, and how to introduce an inactivating mutation within the genes or promoters that control the genes. [0071] The vectors of an aspect of the invention may further comprise at least one expression cassette, wherein the expression cassette comprises one or more transgenes, wherein the one or more transgenes comprise (a) at least one therapeutic gene, (b) at least one reporter gene, or (c) at least one therapeutic gene and at least one reporter gene. The transgene may be any suitable transgene. [0072] The therapeutic gene may be any suitable therapeutic gene. In an aspect of the invention, the at least one therapeutic gene is a dystrophin or an isoform thereof, a sodium voltage-gated channel (SCN), cystic fibrosis transmembrane conductance regulator (CFTR), aromatic l-amino acid decarboxylase (AADC), tyrosine hydroxylase (TH), a GTP cyclohydrolase (GCH), glutamic acid decarboxylase (GAD), complement factor I (CFI), a beta-secretase (BACE), a suppressing siRNA, neprilysin, apolipoprotein E (APOE), nerve growth factor (NGF), or brain derived neurotrophic factor (BDNF). [0073] In an aspect of the invention, the transgene can encode 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), TNFα, IL-4, a neurotrophic factor (e.g., NGF, BDNF, GDNF, NT-3), Ascl1, Nurr1, Lmx1A, Brn2, Myt1l, 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 (“ncRNA(s)”). [0074] In an aspect of the invention, the expression cassette further comprises one or more promoters, wherein each one of the one or more promoters controls expression of one of the one or more transgenes. In an aspect of the invention, the one or more promoters is CAG promoter, a ubiquitin C promoter (UbCp), or a tissue- or cell-specific promoter. In an
University 06350 Leydig 771537 18 aspect of the invention, the one or more is a neuron-specific promoter, a muscle- specific promoter, or a cystic fibrosis promoter. In an aspect of the invention, the one or more promoters is 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), Apo A-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 genes in a defined cell type (e.g., within a liver cell, lung cell, epithelial cell, cardiac cell, neural cell, skeletal muscle cell, embryonic, induced pluripotent, or other stem cell, cancer cell, etc.). Promoters for use in sensory neurons include TRPV1, CGRP, and NF200. In an aspect of the invention, the one or more promoters can be an inducible promoter (e.g., TRE3G combined with rtTA3G expression from a separate promoter in LAT or other inducible promoters as are known in the art). In an aspect of the invention, the one or more promoters can be a constitutive mammalian and viral promoter, such as are known in the art (e.g., SV40, CMV, CAG, EF1α, UbC, RSV, β-actin, PGK, and the like). [0075] The at least one reporter gene may be any suitable reporter gene. In an aspect of the invention, the at least one reporter gene is a fluorescent reporter gene. Any suitable reporter gene may be used. In an aspect of the invention, the at least one reporter gene is ZsGreen (ZsG). In an aspect of the invention, the at least one reporter gene is firefly luciferase (fLuc). The at least one reporter gene can be under the control of any suitable promoter. In an aspect of the invention, the at least one reporter gene is under control of a CAG promoter. [0076] In addition to the at least one promoters, the expression cassette can comprise additional regulatory elements. For example, the expression cassettes can include one or more sites for binding of microRNA. [0077] In an aspect of the invention, the one or more transgenes can be monocistronic (i.e., encoding a single protein or polypeptide) or polycistronic (i.e., encoding multiple proteins or polypeptides). Moreover all or part of the transcribed portion of the transgene also can encode non-translated RNA, such as siRNA or miRNA, for example. In an aspect of the invention, the one or more transgenes can comprise multiple separate monocistronic or polycistronic transgene units (two separate transgene units but possibly more (e.g., three,
University 06350 Leydig 771537 19 four, five, or more separate units)), each with its own respective promoter, translated sequences, or non-translated RNA sequences, and other regulatory elements. [0078] In an aspect of the invention, the insulator sequence comprising at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the LAT locus of the vector. In an aspect of the invention, the insulator sequence comprising at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the LAT locus of the vector and further comprises one or more sequences that encode gene CTRL1. In an aspect of the invention, the insulator sequence comprising the at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the LAT locus of the vector and further comprises one or more sequences that encode gene CTRL2. In an aspect of the invention, the insulator sequence comprising the at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the LAT locus of the vector and further comprises one or more sequences that encode gene CTRL1 and CTRL2. CTRL1 and CTRL2 are insulator sequences native to the LAT locus of HSV. [0079] In an aspect of the invention, the vector further comprises (e.g., retains) one or more sequences that encode enhancer-like LATP2. LATP2 is an enhancer element native to the LAT locus of HSV. In an aspect of the invention, the vector further comprises (e.g., retains) one or more sequences that encode enhancer-like LAP2. LAP2 is another enhancer element native to the LAT locus of HSV. [0080] In an aspect of the invention, the insulator sequence comprising the at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the ICP4 locus of the vector. In an aspect of the invention, the insulator sequence comprising the at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the ICP4 locus of the vector and further comprises one or more sequences that encode gene CTRS3. In an aspect of the invention, the insulator sequence comprising the at least one of SEQ ID NOs: 1-4 (or a sequence with at least 95% identity to any of SEQ ID NOs: 1-4) is positioned in the ICP4 locus of the vector, comprises one or more sequences that encode gene CTRS3, and does not comprise one or more sequences that encode gene CTRS1/2 and/or CTRL2. [0081] In an aspect of the invention, wherein the vector either (a) does not comprise one or more sequences that encode gene ICP22 or (b) comprises one or more sequences that
University 06350 Leydig 771537 20 encode one or more ICP22 genes, with each that encodes the one or more ICP22 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP22 genes. The vectors of an aspect of the invention optionally do not express HSV genes ICP22. There are several ways of modifying HSV so that ICP22 is not expressed. The vector (1) may not comprise one or more sequences that encode ICP22, (2) may comprise one or more sequences that encode ICP22, however, each sequence that encodes ICP22 comprises an inactivating mutation within the sequence, and/or (3) may comprise an inactivating mutation within the promoter region that controls expression of ICP22. It is known by one skilled in the art how to use routine methods to delete all or part of ICP22 gene, and how to introduce an inactivating mutation within ICP22 gene or the promoter(s) that control ICP22 gene. [0082] In an aspect of the invention, the vector is JΔNI8L-ZsG/fLuc as seen in Figure 1A. JΔNI8L-ZsG/fLuc comprises fLuc, and ZsG in the LAT locus. JΔNI8L-ZsG/fLuc further comprises CTRL2, CTRL1 and LATP2 in the LAT locus. JΔNI8L-ZsG/fLuc also has mCherry, CTRS3, and a UbCp promoter in the ICP4 locus. [0083] In an aspect of the invention, the vector is JΔNI84-ZsG/fLuc as seen in Figure 1B. JΔNI84-ZsG/fLuc comprises fLuc, and ZsG in the ICP4 locus. JΔNI84-ZsG/fLuc further comprises CTRS3 in the ICP4 locus. [0084] In an aspect of the invention, the vector is JΔNI84-ZsG/fLuc-2tR or JΔNI8L- ZsG/fLuc-2tR as seen in Figure 9B. JΔNI84-ZsG/fLuc-2tR comprises ZsG, fLuc, and an insulator sequence comprising two copies of SEQ ID NO: 1 in the ICP4 locus. JΔNI8L- ZsG/fLuc-2tR comprises ZsG, fLuc, and an insulator sequence comprising two copies of SEQ ID NO: 1 in the LAT locus. JΔNI8L-ZsG/fLuc-2tR also has CTRL2, CTRL1 and LAPT2 flanking the copies of SEQ ID NO: 1. [0085] In an aspect of the invention, the vector is JΔNI84-ZsG/fLuc-2tR-SMAR or JΔNI8L-ZsG/fLuc-2tR-SMAR as seen in Figure 9C. JΔNI84-ZsG/fLuc-2tR-SMAR comprises an insulator sequence comprising two copies of SEQ ID NO: 1 and one copy of SEQ ID NO: 4, ZsG, and fLuc in the ICP4 locus. SEQ ID NO: 4 is located between the fLuc stop codon and the BGH polyA. JΔNI8L-ZsG/fLuc-2tR-SMAR comprises an insulator sequence comprising two copies of SEQ ID NO: 1 and one copy of SEQ ID NO: 4, ZsG, and fLuc in the LAT locus.
University 06350 Leydig 771537 21 [0086] In an aspect of the invention, the vector further comprises a “self-cleaving” sequence. In an aspect of the invention, the “self-cleaving” sequence is a “self-cleaving” 2A peptide. “Self-cleaving” 2A peptides are described, for example, in Liu, et al., Sci. Rep., 7(1): 2193 (2017), and Szymczak, et al., Nature Biotechnol., 22(5): 589-594 (2004). 2A peptides are viral oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome. Without being bound to a particular theory or mechanism, it is believed that including a self- cleaving peptide will allow co-expression of multiple gene cassettes and creation of single polypeptides at the desired ratio. In an aspect of the invention, the self-cleaving peptide is a porcine teschovirus-12A (P2A) amino acid sequence, equine rhinitis A virus (E2A) amino acid sequence, thosea asigna virus 2A (T2A) amino acid sequence, or foot-and-mouth disease virus (F2A) amino acid sequence. In an aspect of the invention, the self-cleaving peptide has an amino acid sequence comprising, consisting, or consisting essentially of, the amino acid sequence of (T2A). Compositions [0087] An aspect of the invention provides pharmaceutical compositions comprising the vector of an aspect of the invention, and a pharmaceutically acceptable carrier. [0088] The pharmaceutically acceptable carrier of the composition can be any suitable pharmaceutically acceptable carrier for the vector. The carrier typically will be liquid, but also can be solid, or a combination of liquid and solid components. The carrier is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular vector and the particular method used to administer the composition. The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and/or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of an aspect of the invention. The following formulations and methods are merely exemplary and are in no way limiting. [0089] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending
University 06350 Leydig 771537 22 agents, solubilizers, thickening agents, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [0090] In addition, the composition can comprise additional therapeutic or biologically- active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to up-regulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders. Methods [0091] An aspect of the invention provides methods for administering one or more transgenes into a cell in a subject comprising providing the vector of an aspect of the invention, or the pharmaceutical composition of an aspect of the invention, to the subject. [0092] A “subject” as used herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non- human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys. [0093] The vector of an aspect of the invention can be administered in any suitable manner. For example, orally, intranasally, or as an injection (e.g., intravenous, intramuscular, intradermal, subcutaneous, or intra-tumoral). [0094] The vector of an aspect of the invention provides long-term and persistent expression of a transgene. In an aspect of the invention, the vector is capable of expression of the one or more transgenes in a cell for at least about 7 days (e.g., at least about 14 days, at
University 06350 Leydig 771537 23 least about 21 days, at least about 28 days, at least about 35 days, at least about 42 days, at least about 49, days at least about 56 days, at least about 63 days, at least about 70 days, at least about 3 months, at least about 6 months, at least about 9 months, or at least about 12 months). [0095] The vector may be used in any suitable cell. In an aspect of the invention, the cell is an exocrine secretory cell (e.g., glandular cell, such as salivary gland cell, mammary gland cell, sweat gland cell, digestive gland cell, etc.), hormone secreting gland cell (e.g., pituitary cell, thyroid cell, parathyroid cell, adrenal cell, etc.), ectoderm-derived cell (e.g., keratinizing epithelial cell (e.g., making up the skin and hair), wet stratified barrier epithelial cell (e.g., of the cornea, tongue, oral cavity, gastrointestinal tract, urethra, vagina, etc.), cell of the nervous system (e.g., peripheral and central neurons, glia, etc.), mesoderm-derived cell, cell of an internal organs (such as kidney, liver, pancreas, heart, lung), bone marrow cell, and cancerous cell either within tumors or otherwise. In an aspect of the invention, the cell is a liver cell, lung cell, epithelial cell, cardiac cell, muscle cell, stem cell, or neuronal cell, ciliary cell. In an aspect of the invention, the cell is an epithelial cell, a muscle cell, a neuronal cell, or a ciliary cell. In an aspect of the invention, the cell is an epithelial bronchial cell. In an aspect of the invention, the cell is an epithelial nasal cell. In an aspect of the invention, the cell is a cardiac muscle cell. [0096] Aspects, including embodiments, of the subject matter described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1- 25 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: [0097] (1) A recombinant HSV vector comprising: (a) an insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in an intergenic region, a LAT locus, or an ICP4 locus of the vector;
University 06350 Leydig 771537 24 (b) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP0 or (b) comprises one or more sequences that encode one or more ICP0 genes, with each sequence that encodes the one or more ICP0 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP0 genes; (c) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP4 or (b) comprises one or more sequences that encode one or more ICP4 genes, with each sequence that encodes the one or more ICP4 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP4 genes; (d) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP27 or (b) comprises one or more sequences that encode one or more ICP27 genes, with each sequence that encodes the one or more ICP27 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP27 genes; (e) wherein the vector either (a) does not comprise one or more sequences that encode gene ICP47 or (b) comprises one or more sequences that encode one or more ICP47 genes, with each sequence that encodes the one or more ICP47 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP47 genes; (f) wherein the vector either (a) does not comprise one or more sequences that encode internal repeat (joint) region genes or (b) comprises one or more sequences that encode one or more internal repeat (joint) region genes, with each sequence that encodes the one or more internal repeat (joint) region genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more internal repeat (joint) region genes; and (g) wherein the vector either (a) does not comprise one or more sequences that encode gene virion host shut-off (UL41) or (b) comprises one or more sequences that encode one or more UL41 genes, with each sequence that encodes the one or more UL41 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more UL41 genes.
University 06350 Leydig 771537 25 [0098] (2) The vector of aspect 1, further at least one expression cassette, wherein the expression cassette comprises one or more transgenes, wherein the one or more transgenes comprise (a) at least one therapeutic gene, (b) at least one reporter gene, or (c) at least one therapeutic gene and at least one reporter gene. [0099] (3) The vector of aspect 2, wherein the at least one therapeutic gene is a dystrophin or an isoform thereof, a sodium voltage-gated channel (SCN), cystic fibrosis transmembrane conductance regulator (CFTR), aromatic l-amino acid decarboxylase (AADC), tyrosine hydroxylase (TH), a GTP cyclohydrolase (GCH), glutamic acid decarboxylase (GAD), complement factor I (CFI), a beta-secretase (BACE), a suppressing siRNA, neprilysin, apolipoprotein E (APOE), nerve growth factor (NGF), or brain derived neurotrophic factor (BDNF). [0100] (4) The vector of aspect 2 or 3, wherein the expression cassette further comprises one or more promoters, wherein each one of the one or more promoters controls expression of one of the one or more transgenes. [0101] (5) The vector of aspect 4, wherein the one or more promoters is CAG promoter, a ubiquitin C promoter (UbCp), or a tissue- or cell-specific promoter. [0102] (6) The vector of aspect 5, wherein the one or more promoters is a neuron-specific promoter, a muscle-specific promoter, or a cystic fibrosis promoter. [0103] (7) The vector of any one of aspects 2-6, wherein the at least one reporter gene is a fluorescent reporter gene. [0104] (8) The vector of any one of aspects 2-7, wherein the at least one reporter gene is ZsGreen (ZsG) or firefly luciferase (fLuc). [0105] (9) The vector of any one of aspects 2-8, wherein the at least one reporter gene is under control of a CAG promoter. [0106] (10) The vector of any one of aspects 1-9, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in the LAT locus of the vector. [0107] (11) The vector of aspect 10, further comprising one or more sequences that encode gene CTRL1. [0108] (12) The vector of aspect 10 or 11, further comprising one or more sequences that encode gene CTRL2.
University 06350 Leydig 771537 26 [0109] (13) The vector of any one of aspects 1-12, further comprising one or more sequences that encode enhancer-like latency active promoter 2 (LATP2). [0110] (14) The vector of any one of aspects 1-9, wherein the insulator sequence comprising at least 95% identity to at least one of SEQ ID NOs: 1-4 is positioned in the ICP4 locus of the vector. [0111] (15) The vector of aspect 14, further comprising one or more sequences that encode gene CTRS3. [0112] (16) The vector of aspect 14 or 15, wherein the vector does not comprise one or more sequences that encode gene CTRS1/2. [0113] (17) The vector of any one of aspects 14-16, wherein the vector does not comprise one or more sequences that encode gene CTRL2. [0114] (18) The vector of any one of aspects 1-17, wherein the vector either (a) does not comprise one or more sequences that encode gene ICP22 or (b) comprises one or more sequences that encode one or more ICP22 genes, with each sequence that encodes the one or more ICP22 genes comprising an inactivating mutation within the sequence or within the promoter region that controls expression of the one or more ICP22 genes. [0115] (19) A pharmaceutical composition comprising the vector of any one of aspects 1-18, and a pharmaceutically acceptable carrier. [0116] (20) A method for administering one or more transgenes into a cell in a subject comprising: a) providing the vector of any one of aspects 1-18, or the pharmaceutical composition of aspect 19, to the subject. [0117] (21) The method of aspect 20, wherein the vector is capable of expression of the one or more transgenes in a cell for at least 7 days. [0118] (22) The method of aspect 20 or 21, wherein the cell is an epithelial cell, a muscle cell, a neuronal cell, or a ciliary cell. [0119] (23) The method of any one of aspects 20-22, wherein the cell is an epithelial bronchial cell. [0120] (24) The method of any one of aspects 20-22, wherein the cell is an epithelial nasal cell. [0121] (25) The method of any one of aspects 20-22, wherein the cell is a cardiac muscle cell.
University 06350 Leydig 771537 27 [0122] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES Example 1 [0123] This example demonstrates the creation of vectors of an aspect of the invention. [0124] To determine if the genomic location of the transgene cassette and the flanking viral insulators in the ICP4 and LAT locus influence the gene expression profile in different cell types, two vectors were created (see Figures 1A and 1B) carrying the same expression cassette in two different locations of the HSV genome. [0125] Genomic structure of JΔNI8 vectors. In the JΔNI series of vectors, the unique short segment (US) is inverted compared to the “classical” orientation of the HSV genome. The vectors are deleted for the joint region (ΔJOINT), including the ICP47 promoter and translation initiation codon and for the ICP4, ICP27, and ICP0 immediate early (IE) and the virion host shut-off (vhs; UL41) genes (Δ). The envelope glycoprotein gB present the entry- enhancing N/T mutations. In addition, the ICP22 IE is converted to early-expression kinetics (βICP22) by promoter TAATGARAT deletion. [0126] The JΔNI8L-ZsG/fLuc vector contains a transgene cassette expressing ZsGreen (ZsG) and firefly luciferase (fLuc), ZsG/fLuc, under the control of the CAG promoter in the LAT locus. This cassette is surrounded by the viral endogenous insulator sequences (CTRL2, CTRL1, triangles) and the enhancer-like latency active promoter 2 (LATP2). JΔNI8L-ZsG/fLuc contains a second transgene cassette in the ICP4 locus with the Ubiquitin (UbC) driving the expression of the mCherry reporter gene. [0127] In the JΔNI84-ZsG/fLuc, the ZsG/fLuc cassette is located in the ICP4 locus. In this vector, the HSV endogenous insulator elements, CTRS3 is located upstream of the ICP4 locus while the downstream endogenous insulator sequence, CTRS1/2, has been deleted. [0128] JΔNI8L-ZsG/fLuc and JΔNI84-ZsG/fLuc also include a self-cleaving peptide (2A) as shown in Figures 1A and 1B. [0129] Table 1 shows that the viruses can be propagated to approximately the same titers (5 x 10
8 pfu). Table 1 J
ΔNI8 insulator pfu/mla gc/mlb Ratio gc/pfuc
University 06350 Leydig 771537 28 JΔNI8L-ZsG/fLuc 9.10E+08 5.60E+11 615.6
cells.
b Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles.
c Genome copy to pfu ratio. Example 2 [0130] This example demonstrates that vectors of an aspect of the invention are successful in providing transgene expression and survival in a neuronal cell line. This example also demonstrates that at least 2 loci (ICP4 and LAT) can be implemented to deliver therapeutic payloads in a CNS gene therapy strategy.SH-SY5Y cells, a human neuroblastoma cell line, were infected upon differentiation at a MOI of 5,000 gc/cell. Native expression of ZsG transgene was monitored for 21 dpi (see Figure 2A). A luciferase assay was used to monitor the level of fLuc expression. Data were an average of triplicate infections (see Figure 2B). ALAMARBLUE
TM cell viability assay was used to evaluate survival and was performed at several time points (see Figure 2C). Data are shown as percent survival compared to mock infected controls. Data are average of triplicate infections. Figures 2A, 2B, and 2C show that the cassette in the ICP4 locus of a vector of an aspect of the invention is more active in neuronal cultures compared to when the cassette is in the LAT locus. These figures also show that the vectors of an aspect of the invention do not show signs of toxicity. This example further demonstrates that the ICP4 locus is a successful location for gene therapy applications targeting neurons and that the upstream viral insulator and CTRS3 showed enhanced protection activity over the viral insulators flanking the LAT locus. Example 3 [0131] This example demonstrates that vectors of an aspect of the invention are successful in providing in vivo expression of fLuc and ZsG in mouse hippocampus. [0132] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc and JΔNI8L- ZsG/fLuc vectors at MOI of 8*10
8 gc/mouse. Figure 3A shows representative images of mice. Figure 3B shows the level of bioluminescent (BLI) signal in the mice following
University 06350 Leydig 771537 29 injections. Data are average of 6 mice per and are expressed in photons per seconds (p/s). Figures 3A-3B show that long term expression (120 dpi) in mouse brain following intracranial (hippocampus) injection of the vectors of an aspect of the invention. Expression was documented by IVIS
TM 2D Lumina S5 in vivo imaging system following IV luciferin administration. Consistent with the in vitro data, the cassette in the ICP4 locus is stronger, however, both loci are active. [0133] The mice were sacrificed at 5 mpi. Figure 4 shows the identification of ZsG positive cells in JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc injected mouse hippocampi at 5 mpi. From left to right: (column A) DAPI nuclei, (column B) NEUROTRACE
TM (NT) neuronal bodies, (column C) ZsG positive cells, and (columns D-E) DAPI/NT/ ZsG merge panel. Figure 4 (5 mpi) shows that the ICP4 locus showed ZsG-positive cells localized to the neuronal bodies of the CA1, CA2 and CA3 pyramidal cells and axon terminals. In contrast, at this time point, very little ZsG signal was detected from the LAT locus, mainly localized in the CA3 neuronal bodies. Figure 5 (5 mpi) shows consecutive sections of the JΔNI8L- ZsG/fLuc injected mice stained for mCherry (located in the ICP4 locus, Figure 1A) or for ZsG (located in the LAT locus, Figure 1A). Fewer ZsG positive cells were detected compared to mCherry-positive cells especially in the dentate gyrus. Moreover, in the CA3 hippocampal region the ZsG signal did not always colocalize with the mCherry-positive staining. Together these data confirmed that transgene expression was different at the two loci in vitro and in vivo, suggesting favoring the ICP4 locus for transgene expression in neuronal cells, although differential expression in vivo did not reach significance at the late time points. No toxicity to brain tissue was observed as seen previously with this vector backbone (Verlengia, et al., Sci. Rep., 7: 1507 (2017)). Example 4 [0134] This example demonstrates that vectors of an aspect of the invention are successful in providing in vitro expression of fLuc and ZsG in muscle cell culture. This example also demonstrates that the LAT locus and the flanking viral insulators (CTRL1, CTRL2, and LAP2) show enhanced protection activity in muscle cells compared to the viral insulator flanking the ICP4 locus. This example further demonstrates that the LAT locus is a successful location for a gene therapy approach targeting muscle. [0135] C2C12 cells, a mouse myoblast cell line, were infected upon differentiation at a MOI of 5,000 gc/cell. Native expression of ZsG transgene was monitored for 12 dpi (Figure
University 06350 Leydig 771537 30 6A). Luciferase assay was used to monitor the level of fLuc expression (Figure 6B). Data are an average of triplicate infections. cell viability assay was used to evaluate survival at several time points (Figure 6C). Data are shown as percent survival compared to mock infected controls. Data are an average of triplicate infections. [0136] Figures 6A and 6B show that the cells can be differentiated into myotubes using favorable culture conditions. In these cells, the cassette being positioned in the LAT locus was favorable because this vector showed high levels of persistent expression, whereas the cassette in the ICP4 locus was lower. Figure 6C shows that the vectors of an aspect of the invention failed to show toxicity in muscle cells. Example 5 [0137] This example demonstrates that vectors of an aspect of the invention are successful in providing in vivo expression of fLuc and ZsG in the mice hind limb muscles. [0138] This example also demonstrates that the LAT locus and the flanking insulator elements allow for high expression levels of the transgene cassette in vivo up to 1 year post vector delivery. Similarly to the in vitro data, the expression profile of the ICP4 locus in vivo is reduced compared to the LAT locus. [0139] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc or JΔNI8L- ZsG/fLuc vectors into the hind limb muscles at MOI of 1.2*10
9 gc/mouse (approximately equivalent to ~10e6 pfu). Representative images showing the localization of the signal to the mice hind limb muscle are shown in Figure 7A with 1 dpi in the upper panel and 365 dpi in the lower panel. The levels of bioluminescent (BLI) signal in the mice injected with JΔNI84- ZsG/fLuc and JΔNI8L-ZsG/fLuc vectors are shown in Figure 7B. The black line represents the background. Data are an average of 8 mice per group and are expressed in photons per seconds (p/s). Remarkably the amount of expression shown by the IVIS
TM 2D Lumina S5 in vivo imaging system increases over time with both vectors, however, JΔNI8L-ZsG/fLuc had higher expression levels in muscle as reflected in the in vitro test. [0140] Figures 8A and 8B show ZsG distribution upon DI of the mice hind limb muscles with JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc at 365 dpi. Figure 8A shows the area of the muscle where positive ZsG cells are detected. Figure 8B from left to right representative images of (i) DAPI (nuclei), (ii) Dystrophin (DSY) positive cells, (iii) ZsG positive cells, and (iv) DAPI/DYS/ZsG merge panel. Boxes in the upper panels indicate the magnified area as
University 06350 Leydig 771537 31 seen in the lower panels. Figures 8A and 8B further show the in vivo infection and expression in muscle cells as was observed in the in vitro experiment. Example 6 [0141] This example demonstrates the creation of vectors of an aspect of the invention. [0142] To determine if the expression kinetics of the LAT and ICP4 loci can be influenced, additional DNA elements were explored. [0143] JΔNI8 vectors were created that contain insulator sequences in the ICP4 or LAT locus. Schematics of the vectors can be seen in Figures 9A-9C. A control transgene cassette containing the ZsG and fLuc reporter genes under the control of the CAG promoter was used to generate the control vectors JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc by Gateway (GW) reaction (see Figure 9A). The expression cassette described in Figure 9A flanked by the mouse tRNA for glutamine and glycine sequences (SEQ ID NO: 1, 2tRNA, 492 bp) was separated by AT-rich sequences and oriented in opposite direction upstream and downstream of the expression cassette (2tR insulator design), was used to generate JΔNI84-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR by GW reaction (see Figure 9B). The expression cassette described in Figure 9B carrying the matrix attachment region sequence (SEQ ID NO: 4, SMAR, 2 kb), derived from the human β-interferon gene cluster located between the fLuc stop codon and the BGH polyA (2tR-SMAR insulator design), was used to generate JΔNI84- ZsG/fLuc-2tR-SMAR and JΔNI8L-ZsG/fLuc-2tR-SMAR by GW reaction (see Figure 9C). These new vectors include insulator constructs in which the insulators are derived from mouse tRNA genes with and without S/MAR AT-rich sequences. The same two loci act as hosts for this new insulator system. The organization is complex, and the insertions were made in the virus with flanking viral endogenous insulators. Previously, the mammalian insulators driving reporter genes were inserted in two independent intergenic noncoding loci (Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)) that failed to provide long term expression in the brain (Han, et al., J. Virol., 92(17): e00536-18 (2018)). Example 7 [0144] This example demonstrates that vectors of an aspect of the invention express transgenes at high levels during virus replication and that the insulator design of an aspect of the invention does not affect virus growth during production. [0145] Cells from a U2OS 4/27 line were used for viral propagation and were infected at MOI of 0.5 gc/cell and the supernatant were harvested from triplicate wells (n=3) at indicated
University 06350 Leydig 771537 32 time points and titered by qPCR for U
L5 (see Figure 10A). Luciferase expression on U2OS 4/27 cell line at indicated time points (see Figure 10B). Data are presented as average of 3 biological replicates. Table 2 (below) and Figures 10A and 10B show that the vectors of an aspect of the invention can be produced to comparable titers as to vectors shown in Figures 1A and 1B and that the transgenes are expressed at high levels during virus replication. Table 2 J
ΔNI8 insulator pfu/mla gc/mlb Ratio gc/pfuc
a Plaque Forming Unit (pfu) titers were determined by standard plaque assay on U2OS4/27 cells.
b Genome copy titers were determined by qPCR for U
L5; DNA from purified virus particles.
c Genome copy to pfu ratio. [0146] Figures 11A and 11B show the ZsG native expression as evaluated at several time points during virus production. Example 8 [0147] This example demonstrates that the 2tR-SMAR, but not the 2tR, insulator design provides high transgene expression in neuronal cells when located in either the ICP4 and LAT loci. [0148] SH-SY5Y cells, a human neuroblastoma cell line, were infected upon differentiation at a MOI of 5,000 gc/cell with vectors of an aspect of the invention. Figure
University 06350 Leydig 771537 33 12A shows native expression of ZsG at 21 upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L- ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR. Figure 12B show quantification of fLuc activity levels. The 2tR insulator design at the ICP4 and LAT loci does not change the expression levels in neurons compared to the expression levels of the viral insulator alone (with the exception of the first and last time point studied). The 2tR-SMAR insulator design expressed significantly higher levels of transgene at all time points analyzed compared to the vectors JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc in which the expression cassette is only flanked by the viral insulators. Figure 12C shows ALAMARBLUE
TM cell viability assay. This graph shows that the vectors described in Figures 9A, 9B and 9C of an aspect of the invention failed to show toxicity in neuronal cells. [0149] This example demonstrates that the 2tR-SMAR insulator design located in either ICP4 or LAT locus augment transgene expression compared to the viral insulator alone present at these sites. Moreover, no significant difference was observed between the expression levels of JΔNI8L-ZsG/fLuc-2tR-SMAR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors, suggesting that addition of the 2tR-SMAR insulator design at the ICP4 or LAT locus not only enhanced the levels of transgene expression in neurons, but also eliminated the differences observed between these two locations in rdHSV vectors. Example 9 [0150] This example demonstrates that the 2tR-SMAR, but not the 2tR insulator design located in the ICP4 locus, was successful in providing enhanced stable long-term in vivo expression of fLuc and ZsG in the mice brain. [0151] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc, JΔNI84- ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors into the dorsal hippocampus at MOI of 8*10
9 gc/mouse (approximately equivalent to ~10e6 pfu). Representative images of the mice are shown in Figure 13A with 3 dpi in the upper panel and 168 dpi in the lower panel. The levels of bioluminescent (BLI) signal in the mice injected with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors are shown in Figure 13B. The black line represents the background. Data are an average of 6 mice per group and are expressed in photons per seconds (p/s). Remarkably, the amount of expression shown by the IVIS
TM 2D Lumina S5 in vivo imaging system with the 2tR-SMAR insulator design located
University 06350 Leydig 771537 34 in the ICP4 locus is significantly higher up to 12 weeks-post-infection (wpi) compared to the expression levels of JΔNI84-ZsG/fLuc (viral insulator alone). The expression levels tended to remain substantially higher for the remainder of the study. [0152] Figure 14 shows that all three vectors (JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, and JΔNI84-ZsG/fLuc-2tR-SMAR) led to the identification of positive ZsG cells mainly in the CA3 pyramidal cells layer and, to a lesser extent, in the granular cells of the dentate gyrus. The ICP4 locus flanked by the 2tR-SMAR insulator design exhibited higher levels of ZsG expression where well-defined dendrites and axon projections are still visible at 6 mpi. To determine if the cellular insulators inserted in the ICP4 locus can affect rdHSV cell type tropism, the cells were co-stained with NEUROTRACE
TM and observed that in the presence of these insulator designs, the principal cell type expressing the transgene are still neurons. [0153] Figure 15 shows the spread of the signal from the site of injection at 2 wpi and 6 mpi. One section every 300 μm across the hippocampus was analyzed to identify slices presenting ZsG-positive cells. ZsG signal was detected spanning a range of more than 3 mm of the hippocampal thickness in all vectors at 2 wpi, although considerable higher levels of ZsG positive signal was detected from the 2tR-SMAR insulator design. At 6 mpi the abundance of ZsG signal declines for the JΔNI84-ZsG/fLuc (as previously shown) and for the JΔNI84-ZsG/fLuc-2tR vectors. Hippocampal tissue harvested from animals injected with the 2tR-SMAR insulator design (JΔNI84-ZsG/fLuc-2tR-SMAR) still showed vastly higher signal spanning from the rostral-caudal direction of the hippocampus. [0154] Figure 16 shows quantification of fLuc activity in the mice brains up to 11 mpi. These data confirm that the 2tR-SMAR insulator design provides for long-term enhanced transgene expression in the brain. Example 10 [0155] This example demonstrates that the 2tR and 2tR-SMAR insulator designs located in the ICP4 locus are successful in restoring transgene expression at this site in rdHSV vectors in muscle cells. [0156] Cells from C2C12 mouse myoblast cell line were infected 7 days post differentiation at MOI of 5,000 gc/cell with vectors of an aspect of the invention. Figure 17A shows the ZsG native expression upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc- 2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L- ZsG/fLuc-2tR-SMAR at 10 dpi. Taken together, Figures 17A shows that the 2tR and 2tR-
University 06350 Leydig 771537 35 SMAR insulator designs restore expression in the ICP4 locus in vitro and ZsG transgene expression in myotubes is similar for both vectors and have less tissue specificity as compared to the vectors of Figures 1A and 1B. [0157] Luciferase expression was also evaluated at several time points (Figure 17B). Data are shown as average of 3 biological replicates. Figure 17B shows the dramatic improvement in transgene expression with the addition of the 2tR or 2tR-SMAR insulator design in the ICP4 locus in vitro in C2C12 cells compared to JΔNI84-ZsG/fLuc (which is only flanked by the viral insulator) and that the 2tR-SMAR insulator design allows for maximum transgene expression. Flanking the LAT locus with the 2tR insulator design shows a negative effect on transgene expression, while flanking the LAT locus with the 2tR-SMAR design shows a similar expression profile to JΔNI8L-ZsG/fLuc (flanked by only the viral insulators). No differences are observed between the ICP4 and LAT locus in expression profile when the 2tR-SMAR is present. [0158] Cell survival levels were evaluated by ALAMARBLUE
TM cell viability assay at several time points (Figure 17C). Data are shown as percent survival compared to mock infected controls. Data are average of triplicate infections. Figure 17C shows that none of the vectors induced toxicity for non-complementing C2C12 cells following differentiation into myotubes in which the virus cannot replicate. [0159] Figure 18 shows ZsG native expression colocalize with dystrophin expression in differentiate C2C12 cells. C2C12 cells were stained with dystrophin upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L- ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR vectors. ZsG native expression is shown in light grey, and nuclei are stained with DAPI. Figure 18 shows that ZsG and dystrophin are present in the same cells after myotube formation and thus had no effect on dystrophin expression or muscle differentiation. [0160] This example demonstrates that the 2tR insulator design has a negative effect on transgene expression when located in the LAT locus in vitro and that the 2tR-SMAR insulator design has no significant effect on transgene expression compared to the expression profile of the LAT locus flanked only by viral insulators. This example also demonstrates that the 2tR and 2tR-SMAR insulator designs are able to provide enhanced stable long-term in vitro expression of fLuc and ZsG in muscle cells when located in the ICP4 locus. This example further demonstrates that flanking the ICP4 with the 2tR and 2tR-SMAR insulator
University 06350 Leydig 771537 36 designs creates a second suitable location for therapy approaches targeting muscle. In addition, this example demonstrates the possibility to deliver with one single vector two independently regulated transgene cassette to target muscle. Example 11 [0161] This example demonstrates that the 2tR-SMAR insulator design significantly enhances transgene expression at the ICP4 locus compared to the vector flanked only by viral insulators in this region, providing in vivo expression of fLuc and ZsG up to 6 mpi in the mice hind limb muscles. [0162] This example also demonstrates that the 2tR insulator design significantly enhances transgene expression at the ICP4 locus up to 5 wpi upon in vivo delivery to the mice hind limb muscles compared to the vector flanked only by viral insulators in this region. The 2tR insulator design shows a tendency to express higher levels even if not significant for the remaining time course analyzed. [0163] This example further demonstrates that the 2tR and 2tR-SMAR insulator designs have no significant effect in transgene expression in the LAT locus upon in vivo delivery to the mice hind limb muscles. The 2tR follows the expression profile of the LAT locus flanked by only the viral insulators, while the 2tR-SMAR insulator design in the LAT locus shows a tendency to express higher levels, even if not significant, of transgene compared to the vector flanked only by the viral insulators in this region. [0164] This example demonstrates the possibility to deliver with one single vector two independently regulated transgene cassette to target muscle in vivo. [0165] BALB/c mice were injected at 5 weeks with JΔNI8L-ZsG/fLuc, JΔNI8L- ZsG/fLuc-2tR, and JΔNI8L-ZsG/fLuc-2tR-SMAR into hind limb muscles at MOI of 1.2*10
9 gc/mouse (approximately equivalent to ~10e6 pfu). The levels of bioluminescent (BLI) signal are shown in Figure 19A. The black line represents the background. Data are an average of 6 mice per group and are expressed in photons per seconds (p/s). The 2tR and 2tR-SMAR insulator design had no significant effect on transgene expression at the LAT locus. [0166] Figure 19B shows the levels of bioluminescent (BLI) signal upon injection of JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, and JΔNI84-ZsG/fLuc-2tR-SMAR into hind limb muscles at MOI of 1.2*10
9 gc/mouse. Remarkably the amount of expression shown by the
University 06350 Leydig 771537 37 IVIS
TM 2D Lumina S5 in vivo imaging increases over time with the 2tR-SMAR insulator design located in the ICP4 locus. Example 12 [0167] This example demonstrates the creation of vectors of an aspect of the invention. [0168] To determine whether the 2tR-SMAR insulator design also requires the presence of the flanking viral insulators elements present at the ICP4 and LAT loci to protect transgene expression in muscle and neuronal cells, JDNI vectors were created that contain the 2tR- SMAR insulator sequence in an intergenic region of the rdHSV. [0169] A schematic of the vectors can be seen in Figure 20. The fLuc gene was introduced into the vector under the control of the CAG promoter, with or without, 2tR- SMAR insulator design into the UL50-UL51 intergenic region of a dual-reporter, IE gene deficient vector, JΔNI7GFP, via a GW intermediate denoted JΔNI7GFP-GW (Han et al. 2018; Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)). The resulting viruses, JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR, were grown on U2OS 4/27 line for virus propagation. [0170] Table 3 (below) show that the vectors of an aspect of the invention can be produced to comparable titers as to vectors shown in Figures 1A and 1B and that the 2tR_SMAR insulator design located in the UL50-UL51 intergenic region does not affect virus growth during production. [0171] JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR were tested in the differentiated SH-SY5Y and C2C12 cells. Table 3
aque ormng Un t (p u) tters were determned by standard paque assay on U OS / 7 cells.
b Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles.
c Genome copy to pfu ratio. Example 13
University 06350 Leydig 771537 38 [0172] This example demonstrates that the 2tR-SMAR insulator design provides protection in neuronal and muscle cells in vitro independently from the viral insulator present at the ICP4 and LAT locus. [0173] Figure 21A shows a graph quantifying the fLuc activity upon infection (5,000 gc/cell) with JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR in SH-SY5Y human neuronal cells. Luciferase activity was significantly higher at all time points analyzed when the 2tR- SMAR insulator design flanks the CAG-fLuc cassette located in the UL50-UL51 intergenic region. Figure 21B shows ALAMARBLUE
TM cell viability assay. The graph shows that the vectors described in Figure 20 of an aspect of the invention failed to show toxicity in neuronal cells. [0174] Figure 22A shows a graph quantifying the fLuc activity upon infection (5,000 gc/cell) with JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR in C2C12 differentiated mouse myoblasts. Luciferase activity was significantly higher at all time points analyzed when the 2tR-SMAR insulator design flanks the CAG-fLuc cassette located in the UL50- UL51 intergenic region. Figure 22B shows ALAMARBLUE
TM cell viability assay. The graph shows that the vectors descried in Figure 20 of an aspect of the invention failed to show toxicity in muscle cells. Example 14 [0175] This example demonstrates that the LAT locus flanked by the viral insulators (CTRL1, CTRL2, and LAP2) is able to express the 14-kb cDNA encoding the murine version of dystrophin, the defective protein in Duchenne muscular dystrophy (DMD) in vivo in D2.mdx mice. [0176] This example demonstrates the first vector system that is capable of vigorous production of full-length murine dystrophin in vivo in an animal model of DMD. [0177] The engineering of J∆NI7-mDMD has been previously shown (Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)). Briefly, a GW cassette was introduced into the LAT locus between LATP2 and CTRL2, creating J∆NI7-GWL1, and then the cassette was combined with the 16.5-kb insert of a pENTR construct containing the complete mouse dystrophin cDNA between the CAG promoter and the rabbit β-globin polyadenylation region. The resulting vector, J∆NI7-mDMD, was able to express in dystrophin-deficient mdx mouse- derived muscle progenitor cells the full-length dystrophin in vitro (Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)).
University 06350 Leydig 771537 39 [0178] J∆NI7-mDMD was grown on U2OS 4/27 line for virus propagation. Table 4 (below) shows that the vector can be produced to comparable titers as to vectors shown in Figures 1A and 1B and that the mouse full-length cDNA for dystrophin does not affect virus growth during production. [0179] Figure 23 shows the identification of dystrophin positive fibers in the hind limb muscles of DBA wild-type mice injected with PBS and D2.mdx mice injected with either JΔNI7-mDMD (at MOI of 1.2*10
9 gc/mouse) or PBS at 2 wpi. Representative cross section of hind limb muscles (8 sections/animals, 3 animals/group). Groups and treatments are reported on the left. Upper panels show low magnification images (4X); lower panels show higher magnifications of the areas framed in white in the upper panels A; (column A) Nuclei, stained with DAPI; (column B) dystrophin stained positive fibers; (column C) DAPI and dystrophin double-fluorescence. [0180] Figure 24 shows dystrophin distribution in the hind limb muscle of D2.mdx mice upon DI of JΔNI7-mDMD at 2 wpi. A representative cross section of hind limb muscle (8 sections/animals, 3 animals) injected with JΔNI7-mDMD and sacrificed at 2 wpi is shown. Middle panels show low magnification images (4X); upper panels show higher magnifications of the muscle area where abundant DYS transduced fiber are observed (framed in white in the middle panel column A); lower panels show higher magnifications of the muscle area where a clear distinction between DYS transduced and untransduced fibers are observed (framed in white in the middle panel of column A. (Column A) Nuclei, stained with DAPI; (column B) DYS positive fibers; (column C) DAPI and dystrophin double- fluorescence. White * in the bottom panel of column A is indicating the limit between DYS transduced and untransduced fibers. Table 4
q g p y p q y cells.
b Genome copy titers were determined by qPCR for U
L5; DNA from purified virus particles.
c Genome copy to pfu ratio.
University 06350 Leydig 771537 40 [0181] All references, including 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. [0182] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed 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 (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any 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 construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are 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 otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0183] Preferred aspects and embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects and embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-
University 06350 Leydig 771537 41 described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
University 06350 Leydig 771537 28 JΔNI8L-ZsG/fLuc 9.10E+08 5.60E+11 615.6
cells. b Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles. c Genome copy to pfu ratio. Example 2 [0130] This example demonstrates that vectors of an aspect of the invention are successful in providing transgene expression and survival in a neuronal cell line. This example also demonstrates that at least 2 loci (ICP4 and LAT) can be implemented to deliver therapeutic payloads in a CNS gene therapy strategy.SH-SY5Y cells, a human neuroblastoma cell line, were infected upon differentiation at a MOI of 5,000 gc/cell. Native expression of ZsG transgene was monitored for 21 dpi (see Figure 2A). A luciferase assay was used to monitor the level of fLuc expression. Data were an average of triplicate infections (see Figure 2B). ALAMARBLUETM cell viability assay was used to evaluate survival and was performed at several time points (see Figure 2C). Data are shown as percent survival compared to mock infected controls. Data are average of triplicate infections. Figures 2A, 2B, and 2C show that the cassette in the ICP4 locus of a vector of an aspect of the invention is more active in neuronal cultures compared to when the cassette is in the LAT locus. These figures also show that the vectors of an aspect of the invention do not show signs of toxicity. This example further demonstrates that the ICP4 locus is a successful location for gene therapy applications targeting neurons and that the upstream viral insulator and CTRS3 showed enhanced protection activity over the viral insulators flanking the LAT locus. Example 3 [0131] This example demonstrates that vectors of an aspect of the invention are successful in providing in vivo expression of fLuc and ZsG in mouse hippocampus. [0132] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc and JΔNI8L- ZsG/fLuc vectors at MOI of 8*108 gc/mouse. Figure 3A shows representative images of mice. Figure 3B shows the level of bioluminescent (BLI) signal in the mice following
University 06350 Leydig 771537 29 injections. Data are average of 6 mice per and are expressed in photons per seconds (p/s). Figures 3A-3B show that long term expression (120 dpi) in mouse brain following intracranial (hippocampus) injection of the vectors of an aspect of the invention. Expression was documented by IVISTM 2D Lumina S5 in vivo imaging system following IV luciferin administration. Consistent with the in vitro data, the cassette in the ICP4 locus is stronger, however, both loci are active. [0133] The mice were sacrificed at 5 mpi. Figure 4 shows the identification of ZsG positive cells in JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc injected mouse hippocampi at 5 mpi. From left to right: (column A) DAPI nuclei, (column B) NEUROTRACETM (NT) neuronal bodies, (column C) ZsG positive cells, and (columns D-E) DAPI/NT/ ZsG merge panel. Figure 4 (5 mpi) shows that the ICP4 locus showed ZsG-positive cells localized to the neuronal bodies of the CA1, CA2 and CA3 pyramidal cells and axon terminals. In contrast, at this time point, very little ZsG signal was detected from the LAT locus, mainly localized in the CA3 neuronal bodies. Figure 5 (5 mpi) shows consecutive sections of the JΔNI8L- ZsG/fLuc injected mice stained for mCherry (located in the ICP4 locus, Figure 1A) or for ZsG (located in the LAT locus, Figure 1A). Fewer ZsG positive cells were detected compared to mCherry-positive cells especially in the dentate gyrus. Moreover, in the CA3 hippocampal region the ZsG signal did not always colocalize with the mCherry-positive staining. Together these data confirmed that transgene expression was different at the two loci in vitro and in vivo, suggesting favoring the ICP4 locus for transgene expression in neuronal cells, although differential expression in vivo did not reach significance at the late time points. No toxicity to brain tissue was observed as seen previously with this vector backbone (Verlengia, et al., Sci. Rep., 7: 1507 (2017)). Example 4 [0134] This example demonstrates that vectors of an aspect of the invention are successful in providing in vitro expression of fLuc and ZsG in muscle cell culture. This example also demonstrates that the LAT locus and the flanking viral insulators (CTRL1, CTRL2, and LAP2) show enhanced protection activity in muscle cells compared to the viral insulator flanking the ICP4 locus. This example further demonstrates that the LAT locus is a successful location for a gene therapy approach targeting muscle. [0135] C2C12 cells, a mouse myoblast cell line, were infected upon differentiation at a MOI of 5,000 gc/cell. Native expression of ZsG transgene was monitored for 12 dpi (Figure
University 06350 Leydig 771537 30 6A). Luciferase assay was used to monitor the level of fLuc expression (Figure 6B). Data are an average of triplicate infections. cell viability assay was used to evaluate survival at several time points (Figure 6C). Data are shown as percent survival compared to mock infected controls. Data are an average of triplicate infections. [0136] Figures 6A and 6B show that the cells can be differentiated into myotubes using favorable culture conditions. In these cells, the cassette being positioned in the LAT locus was favorable because this vector showed high levels of persistent expression, whereas the cassette in the ICP4 locus was lower. Figure 6C shows that the vectors of an aspect of the invention failed to show toxicity in muscle cells. Example 5 [0137] This example demonstrates that vectors of an aspect of the invention are successful in providing in vivo expression of fLuc and ZsG in the mice hind limb muscles. [0138] This example also demonstrates that the LAT locus and the flanking insulator elements allow for high expression levels of the transgene cassette in vivo up to 1 year post vector delivery. Similarly to the in vitro data, the expression profile of the ICP4 locus in vivo is reduced compared to the LAT locus. [0139] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc or JΔNI8L- ZsG/fLuc vectors into the hind limb muscles at MOI of 1.2*109 gc/mouse (approximately equivalent to ~10e6 pfu). Representative images showing the localization of the signal to the mice hind limb muscle are shown in Figure 7A with 1 dpi in the upper panel and 365 dpi in the lower panel. The levels of bioluminescent (BLI) signal in the mice injected with JΔNI84- ZsG/fLuc and JΔNI8L-ZsG/fLuc vectors are shown in Figure 7B. The black line represents the background. Data are an average of 8 mice per group and are expressed in photons per seconds (p/s). Remarkably the amount of expression shown by the IVISTM 2D Lumina S5 in vivo imaging system increases over time with both vectors, however, JΔNI8L-ZsG/fLuc had higher expression levels in muscle as reflected in the in vitro test. [0140] Figures 8A and 8B show ZsG distribution upon DI of the mice hind limb muscles with JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc at 365 dpi. Figure 8A shows the area of the muscle where positive ZsG cells are detected. Figure 8B from left to right representative images of (i) DAPI (nuclei), (ii) Dystrophin (DSY) positive cells, (iii) ZsG positive cells, and (iv) DAPI/DYS/ZsG merge panel. Boxes in the upper panels indicate the magnified area as
University 06350 Leydig 771537 31 seen in the lower panels. Figures 8A and 8B further show the in vivo infection and expression in muscle cells as was observed in the in vitro experiment. Example 6 [0141] This example demonstrates the creation of vectors of an aspect of the invention. [0142] To determine if the expression kinetics of the LAT and ICP4 loci can be influenced, additional DNA elements were explored. [0143] JΔNI8 vectors were created that contain insulator sequences in the ICP4 or LAT locus. Schematics of the vectors can be seen in Figures 9A-9C. A control transgene cassette containing the ZsG and fLuc reporter genes under the control of the CAG promoter was used to generate the control vectors JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc by Gateway (GW) reaction (see Figure 9A). The expression cassette described in Figure 9A flanked by the mouse tRNA for glutamine and glycine sequences (SEQ ID NO: 1, 2tRNA, 492 bp) was separated by AT-rich sequences and oriented in opposite direction upstream and downstream of the expression cassette (2tR insulator design), was used to generate JΔNI84-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR by GW reaction (see Figure 9B). The expression cassette described in Figure 9B carrying the matrix attachment region sequence (SEQ ID NO: 4, SMAR, 2 kb), derived from the human β-interferon gene cluster located between the fLuc stop codon and the BGH polyA (2tR-SMAR insulator design), was used to generate JΔNI84- ZsG/fLuc-2tR-SMAR and JΔNI8L-ZsG/fLuc-2tR-SMAR by GW reaction (see Figure 9C). These new vectors include insulator constructs in which the insulators are derived from mouse tRNA genes with and without S/MAR AT-rich sequences. The same two loci act as hosts for this new insulator system. The organization is complex, and the insertions were made in the virus with flanking viral endogenous insulators. Previously, the mammalian insulators driving reporter genes were inserted in two independent intergenic noncoding loci (Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)) that failed to provide long term expression in the brain (Han, et al., J. Virol., 92(17): e00536-18 (2018)). Example 7 [0144] This example demonstrates that vectors of an aspect of the invention express transgenes at high levels during virus replication and that the insulator design of an aspect of the invention does not affect virus growth during production. [0145] Cells from a U2OS 4/27 line were used for viral propagation and were infected at MOI of 0.5 gc/cell and the supernatant were harvested from triplicate wells (n=3) at indicated
University 06350 Leydig 771537 32 time points and titered by qPCR for UL5 (see Figure 10A). Luciferase expression on U2OS 4/27 cell line at indicated time points (see Figure 10B). Data are presented as average of 3 biological replicates. Table 2 (below) and Figures 10A and 10B show that the vectors of an aspect of the invention can be produced to comparable titers as to vectors shown in Figures 1A and 1B and that the transgenes are expressed at high levels during virus replication. Table 2 JΔNI8 insulator pfu/mla gc/mlb Ratio gc/pfuc
a Plaque Forming Unit (pfu) titers were determined by standard plaque assay on U2OS4/27 cells. b Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles. c Genome copy to pfu ratio. [0146] Figures 11A and 11B show the ZsG native expression as evaluated at several time points during virus production. Example 8 [0147] This example demonstrates that the 2tR-SMAR, but not the 2tR, insulator design provides high transgene expression in neuronal cells when located in either the ICP4 and LAT loci. [0148] SH-SY5Y cells, a human neuroblastoma cell line, were infected upon differentiation at a MOI of 5,000 gc/cell with vectors of an aspect of the invention. Figure
University 06350 Leydig 771537 33 12A shows native expression of ZsG at 21 upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L- ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR. Figure 12B show quantification of fLuc activity levels. The 2tR insulator design at the ICP4 and LAT loci does not change the expression levels in neurons compared to the expression levels of the viral insulator alone (with the exception of the first and last time point studied). The 2tR-SMAR insulator design expressed significantly higher levels of transgene at all time points analyzed compared to the vectors JΔNI84-ZsG/fLuc and JΔNI8L-ZsG/fLuc in which the expression cassette is only flanked by the viral insulators. Figure 12C shows ALAMARBLUETM cell viability assay. This graph shows that the vectors described in Figures 9A, 9B and 9C of an aspect of the invention failed to show toxicity in neuronal cells. [0149] This example demonstrates that the 2tR-SMAR insulator design located in either ICP4 or LAT locus augment transgene expression compared to the viral insulator alone present at these sites. Moreover, no significant difference was observed between the expression levels of JΔNI8L-ZsG/fLuc-2tR-SMAR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors, suggesting that addition of the 2tR-SMAR insulator design at the ICP4 or LAT locus not only enhanced the levels of transgene expression in neurons, but also eliminated the differences observed between these two locations in rdHSV vectors. Example 9 [0150] This example demonstrates that the 2tR-SMAR, but not the 2tR insulator design located in the ICP4 locus, was successful in providing enhanced stable long-term in vivo expression of fLuc and ZsG in the mice brain. [0151] BALB/c mice were injected at 5 weeks with JΔNI84-ZsG/fLuc, JΔNI84- ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors into the dorsal hippocampus at MOI of 8*109 gc/mouse (approximately equivalent to ~10e6 pfu). Representative images of the mice are shown in Figure 13A with 3 dpi in the upper panel and 168 dpi in the lower panel. The levels of bioluminescent (BLI) signal in the mice injected with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR and JΔNI84-ZsG/fLuc-2tR-SMAR vectors are shown in Figure 13B. The black line represents the background. Data are an average of 6 mice per group and are expressed in photons per seconds (p/s). Remarkably, the amount of expression shown by the IVISTM 2D Lumina S5 in vivo imaging system with the 2tR-SMAR insulator design located
University 06350 Leydig 771537 34 in the ICP4 locus is significantly higher up to 12 weeks-post-infection (wpi) compared to the expression levels of JΔNI84-ZsG/fLuc (viral insulator alone). The expression levels tended to remain substantially higher for the remainder of the study. [0152] Figure 14 shows that all three vectors (JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, and JΔNI84-ZsG/fLuc-2tR-SMAR) led to the identification of positive ZsG cells mainly in the CA3 pyramidal cells layer and, to a lesser extent, in the granular cells of the dentate gyrus. The ICP4 locus flanked by the 2tR-SMAR insulator design exhibited higher levels of ZsG expression where well-defined dendrites and axon projections are still visible at 6 mpi. To determine if the cellular insulators inserted in the ICP4 locus can affect rdHSV cell type tropism, the cells were co-stained with NEUROTRACETM and observed that in the presence of these insulator designs, the principal cell type expressing the transgene are still neurons. [0153] Figure 15 shows the spread of the signal from the site of injection at 2 wpi and 6 mpi. One section every 300 μm across the hippocampus was analyzed to identify slices presenting ZsG-positive cells. ZsG signal was detected spanning a range of more than 3 mm of the hippocampal thickness in all vectors at 2 wpi, although considerable higher levels of ZsG positive signal was detected from the 2tR-SMAR insulator design. At 6 mpi the abundance of ZsG signal declines for the JΔNI84-ZsG/fLuc (as previously shown) and for the JΔNI84-ZsG/fLuc-2tR vectors. Hippocampal tissue harvested from animals injected with the 2tR-SMAR insulator design (JΔNI84-ZsG/fLuc-2tR-SMAR) still showed vastly higher signal spanning from the rostral-caudal direction of the hippocampus. [0154] Figure 16 shows quantification of fLuc activity in the mice brains up to 11 mpi. These data confirm that the 2tR-SMAR insulator design provides for long-term enhanced transgene expression in the brain. Example 10 [0155] This example demonstrates that the 2tR and 2tR-SMAR insulator designs located in the ICP4 locus are successful in restoring transgene expression at this site in rdHSV vectors in muscle cells. [0156] Cells from C2C12 mouse myoblast cell line were infected 7 days post differentiation at MOI of 5,000 gc/cell with vectors of an aspect of the invention. Figure 17A shows the ZsG native expression upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc- 2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L-ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L- ZsG/fLuc-2tR-SMAR at 10 dpi. Taken together, Figures 17A shows that the 2tR and 2tR-
University 06350 Leydig 771537 35 SMAR insulator designs restore expression in the ICP4 locus in vitro and ZsG transgene expression in myotubes is similar for both vectors and have less tissue specificity as compared to the vectors of Figures 1A and 1B. [0157] Luciferase expression was also evaluated at several time points (Figure 17B). Data are shown as average of 3 biological replicates. Figure 17B shows the dramatic improvement in transgene expression with the addition of the 2tR or 2tR-SMAR insulator design in the ICP4 locus in vitro in C2C12 cells compared to JΔNI84-ZsG/fLuc (which is only flanked by the viral insulator) and that the 2tR-SMAR insulator design allows for maximum transgene expression. Flanking the LAT locus with the 2tR insulator design shows a negative effect on transgene expression, while flanking the LAT locus with the 2tR-SMAR design shows a similar expression profile to JΔNI8L-ZsG/fLuc (flanked by only the viral insulators). No differences are observed between the ICP4 and LAT locus in expression profile when the 2tR-SMAR is present. [0158] Cell survival levels were evaluated by ALAMARBLUETM cell viability assay at several time points (Figure 17C). Data are shown as percent survival compared to mock infected controls. Data are average of triplicate infections. Figure 17C shows that none of the vectors induced toxicity for non-complementing C2C12 cells following differentiation into myotubes in which the virus cannot replicate. [0159] Figure 18 shows ZsG native expression colocalize with dystrophin expression in differentiate C2C12 cells. C2C12 cells were stained with dystrophin upon infection with JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, JΔNI84-ZsG/fLuc-2tR-SMAR, JΔNI8L- ZsG/fLuc, JΔNI8L-ZsG/fLuc-2tR and JΔNI8L-ZsG/fLuc-2tR-SMAR vectors. ZsG native expression is shown in light grey, and nuclei are stained with DAPI. Figure 18 shows that ZsG and dystrophin are present in the same cells after myotube formation and thus had no effect on dystrophin expression or muscle differentiation. [0160] This example demonstrates that the 2tR insulator design has a negative effect on transgene expression when located in the LAT locus in vitro and that the 2tR-SMAR insulator design has no significant effect on transgene expression compared to the expression profile of the LAT locus flanked only by viral insulators. This example also demonstrates that the 2tR and 2tR-SMAR insulator designs are able to provide enhanced stable long-term in vitro expression of fLuc and ZsG in muscle cells when located in the ICP4 locus. This example further demonstrates that flanking the ICP4 with the 2tR and 2tR-SMAR insulator
University 06350 Leydig 771537 36 designs creates a second suitable location for therapy approaches targeting muscle. In addition, this example demonstrates the possibility to deliver with one single vector two independently regulated transgene cassette to target muscle. Example 11 [0161] This example demonstrates that the 2tR-SMAR insulator design significantly enhances transgene expression at the ICP4 locus compared to the vector flanked only by viral insulators in this region, providing in vivo expression of fLuc and ZsG up to 6 mpi in the mice hind limb muscles. [0162] This example also demonstrates that the 2tR insulator design significantly enhances transgene expression at the ICP4 locus up to 5 wpi upon in vivo delivery to the mice hind limb muscles compared to the vector flanked only by viral insulators in this region. The 2tR insulator design shows a tendency to express higher levels even if not significant for the remaining time course analyzed. [0163] This example further demonstrates that the 2tR and 2tR-SMAR insulator designs have no significant effect in transgene expression in the LAT locus upon in vivo delivery to the mice hind limb muscles. The 2tR follows the expression profile of the LAT locus flanked by only the viral insulators, while the 2tR-SMAR insulator design in the LAT locus shows a tendency to express higher levels, even if not significant, of transgene compared to the vector flanked only by the viral insulators in this region. [0164] This example demonstrates the possibility to deliver with one single vector two independently regulated transgene cassette to target muscle in vivo. [0165] BALB/c mice were injected at 5 weeks with JΔNI8L-ZsG/fLuc, JΔNI8L- ZsG/fLuc-2tR, and JΔNI8L-ZsG/fLuc-2tR-SMAR into hind limb muscles at MOI of 1.2*109 gc/mouse (approximately equivalent to ~10e6 pfu). The levels of bioluminescent (BLI) signal are shown in Figure 19A. The black line represents the background. Data are an average of 6 mice per group and are expressed in photons per seconds (p/s). The 2tR and 2tR-SMAR insulator design had no significant effect on transgene expression at the LAT locus. [0166] Figure 19B shows the levels of bioluminescent (BLI) signal upon injection of JΔNI84-ZsG/fLuc, JΔNI84-ZsG/fLuc-2tR, and JΔNI84-ZsG/fLuc-2tR-SMAR into hind limb muscles at MOI of 1.2*109 gc/mouse. Remarkably the amount of expression shown by the
University 06350 Leydig 771537 37 IVISTM 2D Lumina S5 in vivo imaging increases over time with the 2tR-SMAR insulator design located in the ICP4 locus. Example 12 [0167] This example demonstrates the creation of vectors of an aspect of the invention. [0168] To determine whether the 2tR-SMAR insulator design also requires the presence of the flanking viral insulators elements present at the ICP4 and LAT loci to protect transgene expression in muscle and neuronal cells, JDNI vectors were created that contain the 2tR- SMAR insulator sequence in an intergenic region of the rdHSV. [0169] A schematic of the vectors can be seen in Figure 20. The fLuc gene was introduced into the vector under the control of the CAG promoter, with or without, 2tR- SMAR insulator design into the UL50-UL51 intergenic region of a dual-reporter, IE gene deficient vector, JΔNI7GFP, via a GW intermediate denoted JΔNI7GFP-GW (Han et al. 2018; Miyagawa, et al., PNAS, 112(13): E1632-E1641 (2015)). The resulting viruses, JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR, were grown on U2OS 4/27 line for virus propagation. [0170] Table 3 (below) show that the vectors of an aspect of the invention can be produced to comparable titers as to vectors shown in Figures 1A and 1B and that the 2tR_SMAR insulator design located in the UL50-UL51 intergenic region does not affect virus growth during production. [0171] JΔNI7GFP-fLuc and JΔNI7GFP-fLuc-2tR-SMAR were tested in the differentiated SH-SY5Y and C2C12 cells. Table 3