Gene therapeutic vector for KSHV-associated diseases

Abstract

Disclosed are compositions and methods for the treatment of KSHV-infected malignancies through administering an AAV gene therapy vector. The AAV vector encodes two copies of the TR sequence, a gene promoter and a suicide gene. The gene therapy vector leads to tumor cell apoptosis and further reactivation of KSHV transactivator to further activate therapeutic genes.

Description

GENE THERAPEUTIC VECTOR FOR KSHV-ASSOCIATED DISEASESI. STATEMENT OF GOVERNMENT SUPPORTThis invention was made with government support under Grant No. R01AI167663, R21CA299587, and R01CA290700 awarded by National Institutes of Health. The government has certain rights in the invention.IL CROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application No. 63 / 691,147, filed on September 5, 2024, which is incorporated herein by reference in its entirety.III. BACKGROUND

[0001] The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by multiple copies of a 0.8 kb terminal repeat (TR) sequence. In KSHV-infected cells, KSHV circular genomes (episomes) are maintained by a viral protein LANA (latency-associated nuclear antigen), which binds to terminal repeat regions of the episomes through C-terminal DNA binding domain and host chromosomes through N-terminal chromatin binding domain. Accordingly, the KSHV TR sequence is essential for maintaining and replicating the infected viral genomes in host cells (i.e., KSHV-associated tumors). Because KSHV-associated tumors express some viral proteins, including LANA, many KSHV laboratories focus on the viral proteins as therapeutic targets; however, these attempts have been unsuccessful. Also, the ability to generate recombinant AAV particles only containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. Therefore, we need a new approach to target KSHV-associated tumors.IV. SUMMARY

[0002] Disclosed are methods and compositions related to a method for enhancing the specificity and efficacy of gene therapy in treating Kaposi's sarcoma-associated herpesvirus (KSHV)-related tumors.

[0003] Disclosed herein are gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9),lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL- 6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP.

[0004] Also disclosed herein are gene therapy vectors of any preceding aspect, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis. In some aspects, the tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV transactivator and / or the KSHV transactivator activates the expression of therapeutic genes.

[0005] In one aspect, disclosed herein are anti-KSHV -infected malignancy therapies comprising the gene therapy vector of any preceding aspect and an anti-herpes antiviral (including, but not limited to ganciclovir, acyclovir, valacyclovir, famciclovir, penciclovir, and valganciclovir. For example, disclosed herein are anti-KSHV -infected malignancy therapies comprising an anti-herpes antiviral (such as, for example, ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir) and a gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9), lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma- associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the genepromoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP

[0006] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders (such as, for example, Kaposi’s sarcoma, human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS-related multicentric Castleman’s disease (MCD), or KSHV- inflammatory cytokine syndrome (KICS)) in a subject, comprising administering to the subject the gene therapy vector and / or anti-KSHV-infected malignancy therapy of any preceding aspect. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or inflammatory disorders associated with KSHV in a subject, comprising administering to the subject a therapeutically effective amount of a gene therapy vector (such as, for example, adenovirus vector, adeno- associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9 lentivirus vector, or mini circle (DNA plasmid)), wherein the gene therapy vector comprises at least two copies of the KSHV terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori- RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or K15.

[0007] Also disclosed in one aspect are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders of any preceding aspect, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis. In some aspects, the tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV transactivator and / or the KSHV transactivator activates the expression of therapeutic genes.

[0008] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders of anypreceding aspect, further comprises administering to the subject a therapeutically effective amount of the gene therapy vector in combination with Suberoylanilide hydroxamic acid (SAHA) or BET inhibitors and / or administering ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir in conjunction with the HSV-1 thymidine kinase gene to increase KSHV viral promoter activity to synergistically induce apoptosis in KSHV- infected tumor cells.V. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0010] Figures 1A and IB show KSHV chromatin modifications. Figure 1A represents KSHV latent chromatin modifications in BCBL-1. The position of indicated histone modification was examined with CUT&RUN analyses. The respective peak heights after CPM normalization are indicated on the left panel. The KSHV ORF map was depicted at the bottom of the panel. The terminal repeat region is zoomed at the right after adjusting the scale of peak ranges. Sequence enrichments seen more than 10 times compared with that of the unique region were underlined. Figure IB represents differential chromatin modifications in KSHV-infected cells. Selected histone modifications were compared among KSHV-infected cells. The cell line name was depicted on the left of the panel. The sequence reads (without CPM normalization) were plotted on the KSHV genome. The H3K27Ac ChlP-seq data for BC1 and BC3 are adapted from SRR9956027 and SRR9956035, respectively.

[0011] Figure 2 shows TR region expresses nascent RNAs. GRO-seq signals aligned to the KSHV genome during latency and upon reactivation. GRO-seq data were obtained from the public data. Sequence reads were mapped to the KSHV genome. The KSHV ORF map was depicted, and the terminal repeat region is zoomed in at the bottom of the panel.

[0012] Figures 3A, 3B, and 3C show LANA and K-Rta protein complexes. Figure 3A represents LANA neighboring protein in iSLK cells. Previously identified LANA neighboring chromatin binding proteins by proximity biotin labeling with recombinant KSHV were visualized with STRING. The line between nodes indicates putative protein interactions based on experimental verification (purple line), curated databases (light blue), text mining (yellow green), coexpression (black line), and protein homologies with other organisms (light purple).Figure 3B represents K-Rta and RNA polymerase II interacting protein on chromatin. Previously identified K-Rta interacting proteins that are recruited to RNAPII by the RIME study were visualized with STRING. The RIME showed the formation of a putative large protein complex containing SWI / SNF and mediator complex. Figure 3C represents predicting protein interactions among LANA and K-Rta complexes during reactivation. Theoretical protein complex interactions in the presence of K-Rta are simulated with STRING. Proteins indicated by blue ovals are those in close proximity to LANA identified by proximity biotin labeling while proteins indicated by red ovals are those interacting with K-Rta identified by the RIME assay. The number of lines between two proteins represents the overall confidence of the putative protein-protein interactions.

[0013] Figures 4A, 4B, 4C, and 4D show an overview of LANA complex recruitment sites on KSHV genome in BCBL-1. Figure 4A shows CUT&RUN LANA-interacting protein recruitment sites were determined by CUT&RUN and depicted on the KSHV genome without CPM normalization. The range of sequence reads is shown on the left of the panel. The KSHV ORF map is depicted at the bottom of the panel. The enrichment of sequence reads at TR over the unique region is presented in Table 1 separately. The position of primers used for Figure 4C is indicated at the bottom of the panel. The same sets of CUT&RUN were also performed with BC-1 cells and presented in Fig. 14. Figure 4B shows zooming at Ori-Lyt and PAN RNA promoter in the unique region. One of the LANA complex-recruited sites at the unique region is zoomed, and positional association among CTCF / SMC1 (purple), H3K27Ac nucleosome, LANA complex recruitment site (blue), and K-Rta binding sites (yellow) is emphasized. The position of the H3K27Ac-nucleosome (red shadow) and CTCF binding sites (green shadow) is also indicated. The association with the frequencies of genomic looing is depicted in Fig. 15 shows the origin of Lyic DNA replication and PAN RNA regions are highlighted at the bottom of the panel. K-Rta ChlP-sequence data are adapted from the public database. Figure 4C shows the nucleosome eviction at the origin of DNA replication during reactivation. TREx-K-Rta BCBL-1 cells were left untreated or treated with doxycycline (1 pg / mL) and TPA (20 ng / mL) for 48 h. CUT&RUN was performed using the H3K4me3 antibody. The percent of input was calculated and shown in the panel. The H3K4me3 non-binding region (control region) was used as a negative control. An unpaired t-test was used for calculating the P-value. **P < 0.05. Figure 4D shows proposing latency-lytic switch model. Through sequence- specific DNA binding, K- Rta recruits the SWI / SNF complex at K-Rta binding sites. The SWI / SNF complex slides / evictsnucleosomes that the LANA complex is tethering. The LANA complex binds the H3K27Ac through direct histone binding or BRD4. The destabilization of the LANA complex and nucleosome eviction facilitates RNAPII elongation, which is stalled at Ori-Lyt and PAN RNA promoter regions.

[0014] Figures 5A, 5B, 5C, and 5D show the terminal repeat does not form a fixed genomic loop with the unique region. Figure 5 A shows position of TR ligated DNA fragments. The genomic looping with TR fragments was examined by isolating sequence reads from previous Hi-C data sets. The ligated DNA fragments with TR elements were aligned to the KSHV genome. The scale of the KSHV genome and ORF maps is shown at the bottom of the panel. A non-adjusted scale with a default setting, which is based on the highest peaks, and the adjusted scale to show peaks in the unique region (bottom panel) are presented. Figure 5B shows changes in looping frequencies by reactivation. Site and frequencies of ligated DNA fragments with TR are shown. The site of genomic loops was conserved, but frequencies of genomic looping with TR were increased. Figure 5C shows normalized tag counts found at the peak. The total number of tags was normalized to 10 million mapped tags. Peaks were identified by findPeaks, HOMER (v4.11). Normalized tag counts around the Ori-Lyt- K5 region (left) and TR region (center) are shown. The ratio of the normalized tag counts around the 26-kbp region to the TR region is also shown in the bar chart (right). Figure 5D shows summary of Figure 4 and 5. A putative LANA / TR nuclear body function as a gene regulatory domain is presented. LANA is accumulated at the TR region with direct DNA bindings that increase the concentration of LANA (blue circle) and interacting proteins such as CHD4 (green square) and BRD4 (gray triangle). Locally concentrated transcription-related proteins facilitate the regulation of lytic gene promoters with LANA.

[0015] Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G display the terminal repeat fragment possesses an enhancer function. Figure 6A shows recombination mediated TR cloning. Schematic diagram of TR fragment cloning. Long primers with homology arms to the end of the unique sequence and TR sequence were used to amplify the pBlueScript plasmid. The amplified DNA fragment was transformed into BAC16 containing GS1783 for recombination. Figure 6B shows confirmation of TR insertion. The EtBr-stained agarose gel is shown. The copy number of TR was determined by an 801 -bp incremental size increase. Figure 6C shows a schematic diagram of the luciferase reporter construct. Luciferase reporters were amplified from the pGL3 vector and cloned into EcoRI sites in two orientations (left and right). Figure 6D shows TRactivates the PAN RNA promoter in an orientation-independent manner. Reporter constructs were transfected into 293FT cells, and luciferase activity was measured 48 h post-transfection. The luciferase value with TRO was normalized as 1 , and fold activation is shown. The standard deviation with triplicated samples is shown. Figure 6E displays TR enhances the K-Rta transactivation function. Indicated reporter constructs (right direction) were co transfected with K-Rta expression plasmid in 293FT cells. Fold activation over TRO with vector control transfection is shown. Standard deviation with triplicated samples is shown as error bars. Figure 6F Diagram and expression of LANA mutant. The LANA acidic domain with acidic repeat domain deletion was constructed for the reporter assay. The position of amino acid deletion and expression in 293FT were shown. Figure 6G shows TR enhances LANA’s transcription function. Indicated reporter constructs ( 1 pg) were co transfected with either full-length LANA expression plasmid (0.5 or 2.0 pg) or LANAAIDR (2.0 pg). Luciferase values were measured 48 h post-transfection. For luciferase reporter assays, error bars with standard deviation for triplicated samples were shown. We performed reporter assays at least three times for each setting, and representing results of one of the replicates are shown. Standard deviation with triplicated samples is shown as error bars.

[0016] Figures 7A, 7B, 7C, and 7D display the terminal repeat fragment possesses enhancer function in KSHV-infected 293FT cells. Figure 7A shows a generation of KSHV-infected cells. The BAC16-Wt KSHV was infected in 293FT cells and selected with hygromycin. The BAC16 stable 293FT cells were stained with the indicated antibody after transfection of the K-Rta expression plasmid. DNA was stained with 4',6-diamidino-2-phenylindole. The scale is shown in the panel. Figure 7B shows immunoblotting. Infection and expression of K-Rta were confirmed by immunoblotting. Indicated antibodies were used for immunoblotting. 0-Actin served as the loading control. The position of protein size markers is indicated at the left of the panels. Figure 7C shows Luciferase assay. Indicated reporter constructs were transfected with either vector control or K-Rta expression plasmid. Luciferase values were measured 48 h after transfection. The luciferase unit with TRO reporter with vector control was designated as 1 , and fold activation is shown. Standard deviation with triplicated samples is shown as error bars. Figure 7D displays the confirmation of transfection efficacies. Images of 293FT and KSHV- infected 293FT cells are shown. RFP-expressing plasmid was cotransfected with reporter and / or K-Rta expression plasmid, and transfection efficacies were examined by RFP signals. Images were captured 48 h after transfection. The scale bar is shown in the left panels.

[0017] Figure 8 displays the design of KS-specific gene therapy vector.

[0018] Figure 9 displays the design of KSHV-associated cancer specific gene therapy vector.Two putative advantages of the design are depicted. KSHV-infected cells are shown in red, and noninfected cells are shown in blue. Two viral DNA binding proteins, KSHV LANA and K-Rta DNA sequence specific binding proteins are utilized to increase specificity of the therapeutic gene expression.

[0019] Figures 10A, 10B, 10C, 10D, 10E, and 10F show the construction of KSHV- infection-specific gene expression cassette. Figure 10A shows a schematic diagram of cloning strategy to generate specific gene therapeutic vector. Plasmid names are depicted in the left panel, and restriction enzyme sites used for cloning were also shown. TR: terminal repeat, ITR: inverted terminal repeat. Figure 10B shows a schematic representation of the regulatory mechanism of the gene cassette. The construct consists of terminal repeats (TR) and Ori RNA promoter (OriP) and the mCardinal fluorescent reporter gene. Figure 10C shows fluorescent and bright field image of the 293 / KSHV and the parental 293 cells. The pAAV-TR2-t?rz'P- mCardinal vector was transfected into 293 / KSHV and the parental 293 cells. Images were taken 48 hours post-transfection. Scales; 200pm.Figure 10D shows mean fluorescence intensity (MFI). The mCardinal signal in 293 / KSHV and the parental 293 cells after the transfection of the pAAV-TR2-Orz'P-mCardinal vector were measured with a Flow cytometer at 2 and 5 days posttransfection. Figure 10E shows the proportion of the mCardinal positive cells. The number of mCardinal expressing cells was determined by the Cy5 channel with the flow cytometry at indicated days after transfection. The relative proportion of mCardinal positive cells was depicted as the number of mCardinal cells at Day 2 as 100%. Figure 10F shows enhanced exogenous gene expression with TR fragments. The pAAV-TR2-(?rzP-mCardinal vector with or without two copies of TR sequences was transfected to the 293 / KSHV cells. Fluorescent and bright field images were taken two days after the transfection. Scales; 300pm.

[0020] Figures 10A, 10B, 10C, 10D, 10E, and 10F display the vector design. Figure 10A shows 4the vector map. The vector was prepared by modifying pAAV. CMV. PI. EGFP. WPRE.bGH vector from Addgene. We deleted entire CMV and EGFP coding sequence and generated pAAV. TR2. Ori promoter vector. We generated specific cloning sites downstream of Ori-RNA promoter and cloned p53, HSV-1 tk, or mCardinal cDNAs. Figure 10B demonstrates the transfection of the pAAV-mCardinal vector. The pAAV.TR2 plasmid was transfected to KSHV infected 293 or a parental cell. The expression of mCardinal is examined underfluorescence microscopy. Bright field and scale are indicated. Figure IOC shows the enhancer function of TR2. Increased mCardinal expression with TR2 is confirmed with KSHV-infected 293 cells. Figure 10D represents the mean fluorescence intensity. The fluorescence intensity was determined for mCardinal positive cells by flow cytometry at indicate days after transfection. Figure 10E shows the mCardinal positive cell percentage. The number of mCardinal expressing cells were determined by flow cytometry and fraction of the positive cells is shown. Figure 10F shows Exogenous p53 expression and poly(A) selection. The poly(A) sites were modified by including WSPR motif or by using S V40 poly(A) sites. The p53 expression was measured by RT-qPCR. The inclusion of WSPR motif stabilizes p53 transcripts and increases expression.

[0021] Figure 11 shows the schematic diagram of the SQ injection model to evaluate tumor growth inhibition with direct targeting approach.

[0022] Figure 12 represents the schematic diagram of the SQ injection model to evaluate tumor growth inhibition with indirect targeting approach.

[0023] Figure 13 represents a schematic diagram demonstrating specificity to KSHV etiology in xenograft model.

[0024] Figures 14A and 14B show an overview of LANA complex recruitment sites on KSHV genome in BC1. Figure 14A shows CUT&RUN LANA interacting protein recruitment sites and histone modifications were determined by CUT&RUN and depicted on the KSHV genome without CPM normalization. The range of sequence reads is shown on the left of the panel. The KSHV ORF map is depicted at the bottom of the panel. Figure 14 B shows zooming on Ori-Lyt and PAN RNA promoter in the unique region. One of the LANA complexes recruited sites at the unique region is zoomed, and positional association among CTCF / SMC1, H3K27Ac nucleosome, LANA complex recruitment site is shown. The LANA complex recruitment sites, position of CTCF / SMC1 binding sites, poised RNAPII, and H3K27AC- necleosome were conserved with BCBL-1 (Figure 5).

[0025] Figure 15 shows the transcription regulatory domain at Ori-Lyt to PAN RNA region. Association of paused RNA polymerase II location and local genomic looping is shown by combining Hi-C data with CUT&RUN data sets. Heat map demonstrates frequencies of DNA ligation identified in Capture Hi-C in TRExBCBL-1. Two other PEL cell lines, BC1 and BC3 have very similar transcription regulatory domain formations. Ori-Lyt region forms transcriptional regulatory domain with PAN RNA promoter and genomic domain is insulated by CTCF bindings.

[0026] Figure 16 represents the Luciferase reporter assay. Increased number of TR reporter was co-transfected with K-Rta expression plasmid and examined association between TR copy and enhancer potency. Fold activation over no TR containing reporter plasmid with vector control is shown. Luciferase value with TRO reporter with vector control was normalized as 1.

[0027] Figures 17A, 17B, 17C, 17D, 17E, and 17F shows activation of TR2-OriP promoter in KSHV infected cells. Figure 17A shows a schematic diagram of the iodixanol gradient ultracentrifugation for AAVs isolation. The fraction with 40% iodixanol contains AAVs. Figure 17B shows SDS-PAGE gels. The 40% iodixanol layer was fractionated from bottom to top 1 to 6. Coomassie staining shows the AAV capsid protein, VP1, VP2, and VP3. The fractions 1 to 4 for AAV8-TR2 OriP mCardinal were pooled, concentrated, measured DNA copies, and used for the following studies. The molecular size marker is indicated on the left side of the gel. Figure 17C shows transmission electron microscopy (TEM) The representative TEM images for the AAV8-TR2-OriP-mCardinal are shown. Putative empty capsids are marked with an arrow. Figure 17D shows fluorescent and bright- field images. The 293 / KSHV and the parental 293 cells were infected with AAV8-TR2-6>rz'P-mCardinal and images were taken 72 hours posttransduction. Scales; 100 pm Figure 17E shows mean fluorescence intensity (MFI) of the mCardinal signal and AAV genome copies. MFI was measured with a flow cytometer, and AAV DNA copies in transduced cells were determined by qPCR. Relative AAV DNA levels were measured at the indicated days after infection. GAPDH coding sequence was used for internal control. Data was analyzed using a two-sided unpaired Student’s t-test and shown as mean ± SD. Figure 17F shows relative abundance of AAV DNA copies. AAV DNA copies were measured by qPCR and compared between the 293 / KSHV and the parental 293 cells. Twenty-four hours after AAV-mCardinal transduction in 293 / KSHV cells was designated as 1. The GAPDH coding sequence was used as an internal control. Data was analyzed using a two-sided unpaired Student's t-test and shown as mean ± SD.

[0028] Figures 18A, 18B, 18C, 18D, and 18E show inhibition of cell growth by AAV8-TR2- OriP-TK in KSHV infection- specific manner. Figure 18A shows 293 cell growth. 293 cells or KSHV -infected 293 cells were seeded in 12 well plates and transduced with AAV8-TR2-Ori P- TK. Cells were treated with mock, GCV (5 pM), or a combination of GCV and OTX015 (200 nM). Live 293 cells were counted every day for three days. The total number of cells in each well was counted in triplicate, and a bar graph was generated with mean ± SD. Figure 18B shows a bar chart with or without A A V8-TR2-(OriP-TK transduction in 293 cells. Live 293 and293 / KSHV cells at Day 3 with or without AAV8-TR2-OriP-TK are shown in bar charts. Data was analyzed using a two-sided unpaired Student’s t-test and shown as mean ± SD. Figure 18C shows iSLK cells growth. iSLK cells or KSHV-infected iSLK cells were seeded in 12 well plates and transduced with AAV8-TR2-OriP-TK. Cells were treated with mock, GCV (5 pM), or GCV and OTX015 (200 nM). The total number of cells in each well was counted in triplicate, and a bar graph was generated with mean ± SD. Figure 18D shows a bar chart with or without AAV8-TR2-OriP-TK transduction in iSLK cells. Live iSLK and iSLK / KSHV cells at Day 3 with or without AAV8-TR2-OriP-TK are shown in bar charts. Data was analyzed using a two- sided unpaired Student's t-test and shown as mean ± SD. Figure 18E shows fluorescent and bright field images. iSLK cells with or without KSHV infection were transduced with AAV8- TR2-OriP-TK. Cells were treated with the indicated drug combination. Images were taken three days after treatment of OTX015 (200 nM) or SAHA (1 pm). Scales; 200pm

[0029] Figures 19A, 19B, 19C, 19D, and 19E show AAV8-TR2-OriP-TK selectively Inhibits KSHV-infected ECFCs growth. Figure 19A shows a schematic diagram of ECFCs differentiation from iPSCs. iPSC differentiation was induced by bFGF, BMP4, and VEGF165 in the Stemline II media, followed by EGF-1I culture. Figure 19B shows relative gene expression of iPSCs and ECFCs (Day 26). The CD34, Prox-1, Flt-4, and LYVE-1 genes were utilized as differentiation markers for ECFCs, while the Oct3 / 4, Nanog, and Sox2 genes were employed as reprogramming markers. 18S was used for internal control. Data was analyzed using a two-sided unpaired Student's t-test and shown as mean ± SD. Figure 19C shows fluorescent and bright field cell images, r.219 KSHV (MOI = 1) was infected to differentiating cells at 12 days postinduction of iPSC differentiation or parental iPSCs. Images were taken 14 days after infection. Figure 19D shows fluorescent and bright field cell images. ECFCs or KSHV-infected ECFCs transduced with AAV8-TR2-Ori P-TK were treated with GCV (5 pM), and images were taken three days after GCV treatment. Figure 19E shows cell growth. ECFCs or KSHV-infected ECFCs were seeded in 6 well plates and transduced with AAV8-TR2-Ori P-TK. The following day, cells were treated with DMSO or GCV (5 pM). Live ECFCs were counted daily for three consecutive days. The total number of cells in each well was counted in triplicate, and a bar graph was generated with mean ± SD.

[0030] Figures 20A, 20B, 20C, 20E, and 20E show AAV8-TR2-Ori P-TK prevents KSHV reactivation and re-infections. Figure 20A shows fluorescence and bright field cell images. iSLK / KSHV cells were treated as indicated, and the cells were imaged with fluorescencemicroscopy at 48 hours post-stimulation. AAV8-TR2-OriP-TK transduction with GCV treatment resulted in a decrease in the RFP signal, indicating reduced PAN-RNA promoter activation. Figure 20B shows viral gene expression. PAN-RNA or K8.1 transcripts were measured by RT-qPCR with specific primer pairs. Transcripts were normalized with 18S rRNA. The relative transcripts in AAV8-TR2-OriP-TK transduced without GCV were normalized as 1. Data was analyzed using a two-sided unpaired Student's t-test and shown as mean ± SD. Figure 20C shows capsidated viral DNA copies. The KSHV virion copy number with or without reactivation in the presence or absence of GCV (5 pM ) was measured with qPCR. KSHV virions were collected from the culture supernatant 4 days post-reactivation. Data were analyzed using a two-sided unpaired Student's t-test and presented as mean ± SD. Figure 20D shows KSHV infection in freshly prepared iSLK cells. The KSHV infection with reactivated virus was evaluated by infecting freshly prepared iSLK cells. 1 ml culture supernatant 4 days postreactivation in the presence or absence of GCV (5 pM ) was mixed with freshly prepared iSLK cells. Images were taken two days after infection. Figure 20E shows flow cytometry.Frequencies of KSHV infection were measured with flow cytometry. Data was analyzed using a two-sided unpaired Student's t-test and shown as mean ± SD.

[0031] Figures 21A, 21B, 21C, 21D, and 21E show bystander effects and transcription profiles. Figure 21A shows a schematic diagram of study design for the bystander effect. Two types of KSHV-infected iSLK cells were prepared (Green and Red), and AAV8-TR2-OriP-TK was transduced only to EGFP -positive cells. Green and red (mCherry-positive) cells were mixed at a 1:1 ratio (10A5cells each) and cultured for four days in the presence of GCV (5 pM). Bystander effects were assessed with the viability of red cells. Figure 2 IB shows fluorescence cell images. Arrows indicate dead red cells determined by cell morphology and fractured nucleus. Images were taken four days after the mixture of iSLK / KSHV (green) and iSLK / mCherry-KSHV (red) cells Figure 21C shows principle component analyses. Total RNA sequence was performed with iSLK / KSHV and 293 / KSHV cells. Cells were treated as indicated in the panel. Figure 21D shows pathway analyses. Cellular signaling pathways that are significantly altered in GCV with AAV8-TR2-OriP-TK transduction were shown. The results indicated a strong induction of DNA damage responses with subsequent induction of cell apoptosis. Figure 21E shows immunofluorescence assays. AAV8-TR2-OriP-TK transduced iSLK / KSHV cells were stained with antibodies specific to cleaved caspase 3 and rabbit-647secondary antibody. Cleaved caspase 3 expression in iSLK / KSHV cells under GCV (5 pM ) treatment was confirmed with immune staining.[0032 J Figures 22A, 22B, 22C, 22D, 22E, and 22F showAAV8-TR2-OriP-TK inhibits tumor growth in a xenograft model. Figure 22A shows a schematic diagram of treatment schedules. KSHV-infected iSLK cells were transduced with AAV8-TR2-OriP-TK and xenograft subcutaneous. AAV8-TR2-OriP-TK transduced iSLK cells were implanted into the right hind leg of the mice, while non-transduced iSLK cells were injected into the left hind leg. GCV was administered as indicated. Figure 22B shows a measurement of tumor sizes. Tumor volumes (mm3) were measured every 4 days. Figure 22C shows mouse images at Day 38. Figure 22D shows images of tumor mass extracted from subcutaneous. Figure 22E shows tumor volumes. The weight of tumor mass (mg) was plotted, and treatments were indicated at the bottom of the panel. Figure 22F shows hematoxylin-eosin (H&E) staining and Immunohistochemistry (IHC) staining of Ki-67 in mouse tissue sections. Representative images of IHC staining and EGFP signals found in individual tumors. Scales: 20pm (top and middle), 200pm (bottom).

[0033] Figures 23A, 23B, 23C, 23D and 23E show AAV8-TR2-OriP-TK inhibits tumor growth in a KSHV infection-specific manner in a xenograft model. Figure 23A shows a schematic diagram of treatment schedules. KSHV-infected Capi-1 (SLK cells) and parental SLK cells were transduced with AAV8-TR2-OriP-TK and xenograft subcutaneously to the right and left hind legs, respectively. GCV was administered at 50 mg / kg twice a day for 5 days. Figure 23B shows measurement of tumor sizes. Tumor volumes (mm3) were measured every 3 to 4 days. Figure 23C shows mouse images at Day 40. Figure 23D shows images of tumor mass extracted from subcutaneous. Figure 23E shows tumor volumes. The weight of tumor mass (mg) was plotted, and treatments were indicated at the bottom of the panel.VI. DETAILED DESCRIPTION

[0034] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.A. Definitions

[0035] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0036] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0037] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0038] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0039] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, oraverage increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0040] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0041] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0042] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0043] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0044] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0045] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0046] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0047] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0048] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0049] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0050] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0051] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0052] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate,metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0053] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0054] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0055] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosedare also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.B. Compositions

[0056] Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered in 1994 and is one of the eight human herpesviruses. KSHV is the causative agent of Kaposi’s sarcoma, two human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS -related multicentric Castleman’s disease (MCD), and a more recently described interleukin-6 related disease, KSHV -inflammatory cytokine syndrome (KICS). These highly inflammatory diseases are a leading cause of cancer deaths in AIDS patients in sub-Saharan Africa. KSHV-encoded Latency-associated nuclear antigen (LANA) is frequently identified in KSHV-infected tumor cells. Also, KSHV LANA plays a role in KSHV-mediated tumorigenesis by manipulating cell cycle machinery. Accordingly, we focus on the LANA protein as a therapeutic target to inhibit tumorigenesis and KSHV replication. However, we have yet to develop effective smallmolecule inhibitors for LANA; we need to explore additional directions.

[0057] The KSHV viral genome consists of an approximately 140 kb unique coding region flanked by large copies of high G+C 801 bp terminal repeat (TR). KSHV genomes persist in latently infected cells as circular genomes (episome) via tethering to the host cell chromosomes. During latency, a few latent genes are actively transcribed. Among these latent genes, ORF73 encodes LANA, which plays a crucial role in latent episome replication and maintaining the episome in daughter cells. The TR contains a DNA replication origin, which consists of two LANA-biding sites (LBS): a higher affinity site (LBSl) and a lower affinity site (LBS2) followed by an adjacent 32-bp GC-rich segment. Episome maintenance requires at least two copies of TR (LBS 1 / 2 binding sites). DNA binding induces oligomerization of LANADBD, and a hydrophobic interface between LANA dimer forms the decametric ring and is essential for cooperative DNA binding, hence episome maintenance. These mechanisms substantially increase LANA concentration around the TR, and the TR / LANA complexes can be seen as LANA dots in KSHV-infected cells with immunostaining. The LANA dots (also called LANA nuclear bodies) are used to diagnose KSHV etiology.

[0058] In addition to being a LANA binding sequence, KSHV TR was found to function as a gene enhancer for inducible viral gene promoters. Gene enhancers are a crucial regulatory genomic domain for differential gene expression and are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. The reporter assaysdemonstrated that TR sequences enhanced KSHV inducible promoter activity more than x 100 in 293T cells. Furthermore, TR strongly synergizes with the viral transcription activator, K-Rta, for the transactivation function; this makes the enhancer / promoter pair a very attractive gene element to develop KSHV-infection-specific gene therapy.

[0059] One of the critical barriers of gene therapy is the safe and efficient delivery of genetic material to the target tissues / cells, which is carried out by delivery vehicles. There are two gene therapy vectors: viral and non-viral. Non-viral vectors comprise all the chemical and physical techniques and generally include chemical methods such as cationic liposome and synthetic polymers, or physical methods such as gene gun, electroporation, ultrasound utilization, and magnetoreception. The advantages of non-viral vectors are their costeffectiveness and less induction of immune reaction. However, the most successful gene therapy vectors available today are viral vectors, including retrovirus, adenovirus, lentivirus, and adeno- associated virus (AAV). An advantage of viral vectors is higher delivery efficiency than non- viral methods. Viral genomes are modified for viral vectors by deleting some essential genes, which restricts their replication and allows safer gene delivery to the patients. Among the viruses used for gene therapy vectors, AAV was first discovered in 1965 as a co-infecting agent of adenovirus. The first infectious clone of AAV serotype 2 for human gene therapy was generated in 1982. Since then, AAV serotypes 1-12 and over 100 AAV variants have been identified. AAV is a non-enveloped DNA virus with a genome of approximately 4.8 kbp single- stranded DNA. The coding capacity for the transgene is limited to ~4.7 kbp, but this can be extended by splitting the trans gene sequence into two viruses with co-inf ection. AAVs are naturally replicationdeficient and require a helper virus for replication and dissemination. This self-replication deficiency and their ability to infect both dividing and non-dividing cells stably make them an excellent viral gene therapy vector. AAV gene therapy vectors have received FDA approval for commercialization - Luxturna for retinal dystrophy (NCT00999609) and Zolgensma for spinal muscular atrophy (NCT03306277). The number of FDA-approved AAV-associated drugs is expected to increase because many AAV gene therapy vectors are being tested at later clinical trial stages.

[0060] Indirect gene therapy, a method that converts the prodrug into a lethal (cytotoxic) drug within the tumor cells, demonstrates great promise and is being evaluated in clinical trials. The advantage of an indirect approach is to have an additional step, which makes the therapy adjustable and, therefore, safer for patients. The most studied indirect therapy is based onintroducing the herpes simplex virus thymidine kinase (HSV-TK) gene with ganciclovir (GCV) administration. The HSV-TK initiates the conversion of the antiviral drug, GCV, to a toxic metabolite, GCV-triphosphate, which inhibits DNA synthesis and induces cell apoptosis. The converted cytotoxic compound is also reported to have a bystander effect of killing surrounding non-transduced tumor cells, presumably due to released GCV-triphosphate from dying cells; this minimizes the necessity to transduce the vector to the 100% of tumor cells to be effective.

[0061] Here, we designed and evaluated a KSHV-associated disease-specific gene therapy vector. We first examined the TK / GCV indirect therapy with AAV because KSHV is a herpesvirus, and GCV alone showed some efficacies in preventing KSHV-associated tumor progression in clinics. This report describes the construction and concept of the gene therapy vector and its initial characterization in the tissue culture model and the xenograft mouse model.

[0062] Disclosed herein are gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9), lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene and increasing retention of the DNA vector through LANA protein expressed in KSHV infected tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP.

[0063] Also disclosed herein are gene therapy vectors, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis. In some aspects, the tumor cell apoptosis by the anti-cancer drugs for combination therapies further reactivates KSHV, thereby activating the expression of KSHV transactivator and / or the KSHV transactivator activates the expression of therapeutic genes.1. Delivery of the compositions to cells

[0064] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier. a) Nucleic acid based delivery systems

[0065] Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0066] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as the KSHV gene therapy into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are , for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect nondividing cells. Pox viral vectors are large and have several sites for inserting genes, they arethermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

[0067] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.(1) Retroviral Vectors

[0068] A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer.

[0069] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, andenv genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0070] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.(2) Adenoviral Vectors

[0071] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61: 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediatedendocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650- 655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061- 6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0072] A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.(3) Adeno-asscociated viral vectors

[0073] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4. 1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0074] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0075] Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorproated by reference for material related to the AAV vector.

[0076] The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

[0077] The inserted genes in viral and retroviral usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0078] One of the key steps of gene therapy is the safe and efficient delivery of genetic material to the target tissues / cells, which is carried out by specific delivery vehicles. Currently, there are two types of gene therapy vectors: viral and non-viral. “Non- viral vectors” comprise all the chemical and physical techniques, and generally include either chemical methods such as cationic liposome and synthetic polymers, or physical methods such as gene gun, electroporation, ultrasound utilization and magnetofection. The major advantages of non-viral vectors are their cost-effectiveness and less induction of immune reaction. However, the most successful gene therapy vectors available today are “viral vectors”, including retrovirus, adenovirus, lentivirus, and adeno-associated virus (AAV). A major advantage of viral vectors is higher delivery efficiency than non-viral methods. For viral vectors, viral genomes are modified by deleting some open reading flames, which makes their replication restricted, allowing safer gene delivery to the patients.

[0079] Disclosed herein are gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9), lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL- 6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP. In some aspects, it is understood and herein contemplated that the gene therapy can also be administered as a DNA vaccine rather than comprises in a viral vector.

[0080] Also disclosed herein are gene therapy vectors, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis. Insome aspects, the tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV transactivator and / or the KSHV transactivator activates the expression of therapeutic genes.

[0081] In one aspect, disclosed herein are anti-KSHV -infected malignancy therapies comprising any of the gene therapy vectors disclosed herein and an anti-herpes antiviral (including, but not limited to ganciclovir, acyclovir, valacyclovir, famciclovir, penciclovir, and valganciclovir. For example, disclosed herein are anti-KSHV -infected malignancy therapies comprising an anti-herpes antiviral (such as, for example, ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir) and a gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9), lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma- associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP),

[0082] Disclosed herein is that KSHV TR is not only a LANA binding sequence for KSHV episome maintenance in infected cells, but it also has an essential function as a gene enhancer. The gene enhancer is a genomic sequence that controls (activates) gene promoters for transcription. Based on this discovery, an idea was devised for an innovative KSHV-tumor- specific gene therapy vector with an AAV. The AAV vector was designed to encode two copies of the TR sequence, which will help maintain the therapeutic vector via KSHV LANA. KSHV LANA is only expressed in KSHV-infected tumors. The TR sequence also enhances viral gene promoters and generate vectors that drive tumor suppressor protein HSV-1 thymidine kinase (TK). The strongest KSHV promoter was selected to transcribe the therapeutic genes that are further activated (approximately 50x) in the presence of the KSHV transactivator. The induction of cell apoptosis by the expression of therapeutic gene triggers the KSHV reactivation and, therefore, the KSHV transactivator expression. The FDA-approved drugs such as histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), or BET inhibitor are also known to trigger KSHV reactivation as a combination therapy, further enhances therapeutic gene expression from the vector in KSHV-infected cells, therefore, cancer cell killing.

[0083] Two approaches are used to achieve specific delivery of genes (i) Using a specific promoter, whose activity is increased in cancer cells. Such promoter includes human telomerase reverse transcriptase promoter, (ii) Expressing frequently mutated proapoptotic genes, whose protein function is often compromised only in tumor cells. Among many tumor suppressor genes, mutations of the tumor suppressor p53 (TP53) are the most frequent identified in human tumor cells, and a number of studies have shown that restoring TP53 expression / function can induce apoptotic cell death in these tumor cells. Several direct gene therapy methods / agents targeting TP53 have been clinically examined. For example, Gendicine is a recombinant adenovirus vector expressing wild-type p53 (wild-type TP53), which is designed to treat patients with tumors that have mutated TP53 genes in non-small-lung cancer.

[0084] “Indirect gene therapy”, a method utilizing the conversion of the prodrug into a lethal (cytotoxic) drug within the tumor cells, also demonstrate great promise and is being evaluated in clinical trials. The advantage of an indirect approach is to have an additional step, which makes the therapy adjustable, therefore, safer. The most studied indirect therapy is based on the introduction of herpes simplex virus thymidine kinase (HSV-TK) gene with ganciclovir (GCV) administration. The HSV-TK initiates conversion of the antiviral drug ganciclovir (GCV) to the toxic metabolite GCV-triphosphate, which inhibits DNA synthesis and induces cell apoptosis. The converted cytotoxic compound is also reported to have a bystander effect to kill surrounding non-transduced tumor cells, presumably due to released GCV-triphosphate from dying cells; the effects minimize the necessity to transduce the vector to the 100% tumor cells to be effective. In this application, the TK / GCV is selected, and also compared with the direct target approach (TP53). The reason for the selecting the TK / GCV is that KSHV is a herpesvirus and GCV alone has been shown to control KSHV-associated tumor progression. KSHV also encodes TK gene in their genome. Inhibition of KSHV lytic replication by GCV has a benefit and concluded that small fraction of KSV-infected tumors (primarily latently infected cells) may be spontaneously reactivating. By introducing our AAV TR2-Ori-TK should facilitate cancer cell killing by adding exogenous TK to the endogenous KSHV TK with GCV.

[0085] In KSHV infected cells in patients with KSHV-associated malignancies, KSHV LANA should be expressed in all the tumor cells. Disclosed herein are at least two TR copies for the plasmid maintenance. Thus, having two copies of TR in the therapeutic vector is maintained better in KSHV-infected (LANA-expressing) cells than in non- infected cells. In one aspect, TR can increase viral promoter activity, and the promoter activation was further increased inpresence of KSHV transactivator, K-Rta. Thus, the KSHV reactivation triggered by induction of cell apoptosis, will further increase therapeutic genes expression in the KSHV infected tumor cells, and that in turn prevent KSHV DNA replication by cell death or GCV in the case of the indirect therapy. Therefore, additional benefits are expected with the TK / GCV approach.(4) Large payload viral vectors

[0086] Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et ah, Nature genetics 8: 33-41, 1994;Cotter and Robertson,. Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0087] Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors. b) Non-nudeic add based systems

[0088] The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

[0089] Thus, the compositions can comprise, in addition to the disclosed AAV vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can beadministered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0090] In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAM1NE (G1BCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0091] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other speciifc cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalizationpathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).L0092 J Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.

[0093] Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art. c) In vivo / ex vivo

[0094] As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject=s cells in vivo and / or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

[0095] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.2. Expression systems

[0096] The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. a) Viral Promoters and Enhancers

[0097] Enhancers are a crucial regulator of differential gene expression programs. “Enhancers” are the cis-regulatory sequences determining target genes’ spatiotemporal and quantitative expression. In some aspects, disclosed herein Kaposi’s sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. In one aspect, disclosed herein is a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression. In one aspect, the disclose gene therapy vectors and constructs can comprise at least two, three, four, five, six, seven, or eight or more terminal repeats.

[0098] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindi II E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

[0099] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit.Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers f unction to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0100] The promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0101] In certain embodiments the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

[0102] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0103] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region isthat it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early poly adenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.3.KSHV TR is not only for LANA tethering sequence but also an enhancer for KSHV inducible genes. SHV LANA is essential for maintaining KSHV episomes.

[0104] As noted herein, enhancers are a crucial regulator of differential gene expression programs and are the cis-regulatory sequences that determine target genes' spatiotemporal and quantitative expression. KSHV TR enhances inducible promoter activation as recently demonstrated. Disclosed herein, (i) TR is occupied by coactivator enzymes, (ii) marked with active histone modification, H3K27Ac, (iii) expressing nascent RNAs, and (iv) possesses orientation- independent transcription function. Thus, the KSHV TR is an enhancer. The TR forms phase separate bodies with LANA and encodes multiple specific binding sites as described above; thus the enhancer is designed for LANA to regulate KSHV inducible promoters during evolution; this LANA-specific enhancer design makes the TR as very attractive gene element to make specific gene therapy vector. The reporter assays indeed demonstrated that TR fragments enhanced KSHV inducible promoter activity more than x 100 in 293 T cells. Furthermore, TR also synergizes with viral transcription activator, K-Rta for the transactivation function. The result devised the idea that TR with K-Rta responsive inducible promoter may be used for gene therapy promoters for robust therapeutic gene expression in KSHV infected cells. Ori-RNA promoter was selected out of 80+ lytic gene promoters, which demonstrated the highest promoter activity, and the promoter is one of the 33 KSHV K-Rta targeted promoters.4. Anti-KSHV-infected malignancy therapy

[0105] In one aspect, it is understood and herein contemplated that the disclosed vectors can be used in conjunction with other therapeutics to treat KSHV -infected malignancies. Thus, in one aspect, disclosed herein are anti-KSHV -infected malignancy therapies comprising the gene therapy vector disclosed herein and an anti-herpes antiviral (including, but not limited toganciclovir, acyclovir, valacyclovir, famciclovir, penciclovir, and valganciclovir. For example, disclosed herein are anti-KSHV -infected malignancy therapies comprising an anti-herpes antiviral (such as, for example, ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir) and a gene therapy vectors (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9), lentivirus vector, or mini circle (DNA plasmid)) comprising at least two, three, four, five, six, seven, or eight or more copies of the Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21 ) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori- RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP

[0106] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders (such as, for example, Kaposi’s sarcoma, human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS-related multicentric Castleman’s disease (MCD), or KSHV- inflammatory cytokine syndrome (KICS)) in a subject, comprising administering to the subject the gene therapy vector and / or anti-KSHV-infected malignancy therapy disclosed herein. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders in a subject, comprising administering to the subject a therapeutically effective amount of a gene therapy vector (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector (including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9 lentivirus vector, or mini circle (DNA plasmid)), wherein the gene therapy vector comprises at least two copies of the KSHV terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleosidephophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell. In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or K15.

[0107] It is understood and herein contemplated that the disclosed gene therapy vectors and anti-KSHV infected malignancy therapies can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine 1 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE®(Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clof arabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil- -Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin,GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP,MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, N1NLAR0® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa- 2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUB RAC A® (RucaparibCamsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / orZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrcdizurnab, cemiplimab , CT-011, MK- 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS- 936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA- 4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and 1TIM domains (TiGlT)(such as, for example BMS-986207, OMP- 313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).5. Pharmaceutical carriers / Delivery of pharmaceutical products

[0108] As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0109] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will varyfrom subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0110] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

[0111] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophy sica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors followmore than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers

[0112] The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0113] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0114] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0115] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0116] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous,intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0117] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0118] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0119] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

[0120] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. b) Therapeutic Uses

[0121] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. In one aspect, the anti-herpes antiviral is administered at the pharmaceutically acceptable dosage used in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions,anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303- 357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 pg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.C. Methods of Use

[0122] It is understood and herein contemplated that the gene therapy vectors and anti- KSHV-infected malignancy therapies disclosed herein can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers that is the result of a cell being infected with KSHV (such as, for example, Kaposi’s sarcoma, human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS-related multicentric Castleman’s disease (MCD), B cell lymphoma (including, but not limited to diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, , Burkitt lymphoma, mantel cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma), or KSHV-inflammatory cytokine syndrome (KICS)).

[0123] Accordingly, in one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders (such as, for example, Kaposi’s sarcoma, human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS-related multicentric Castleman’s disease (MCD), or KSHV- inflammatory cytokine syndrome (KICS)) in a subject, comprising administering to the subject the gene therapy vector and / or any of the anti-KSHV malignancy therapies disclosed herein. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders in a subject, comprising administering to the subject a therapeutically effective amount of a gene therapy vector (such as, for example, adenovirus vector, adeno-associated virus (AAV) vector(including, but not limited to AAV serotypes selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9 lentivirus vector, or mini circle (DNA plasmid) or an anti-KSHV malignancy therapy, wherein the gene therapy vector comprises at least two copies of the KSHV terminal repeat (TR) sequence, and a suicide gene (such as, for example, TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, and / or p21) operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell; and wherein the anti-KSHV malignancy therapy comprises the gene therapy vector and an anti-herpes antiviral (including, but not limited to ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir). In some aspects the gene promoter comprises inducible gene promoter or tissue specific gene promoter. In some aspects the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or K15.

[0124] Also disclosed in one aspect are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis. In some aspects, the tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV transactivator and / or the KSHV transactivator activates the expression of therapeutic genes.

[0125] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a KSHV-infected malignancies or disorders, further comprises administering to the subject a therapeutically effective amount of the gene therapy vector in combination with Suberoylanilide hydroxamic acid (SAHA) or BET inhibitors and / or administering ganciclovir (GCV), acyclovir, valacyclovir, famciclovir, penciclovir, and / or valganciclovir in conjunction with the HSV-1 thymidine kinase gene to increase KSHV viral promoter activity to synergistically induce apoptosis in KSHV-infected tumor cells.

[0126] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC- T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine,ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, ARED1A® (Pamidronate Disodium), AR1MIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clof arabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S- Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-AB V, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR- U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, DexrazoxaneHydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERW1NAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil-Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (FluorouraciL -Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRL CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2(Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (DenileukinDiftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUB RAC A® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil-Topical), Topotecan Hydrochloride,Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZEN1O® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XT ANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK- 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS- 936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA- 4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP- 313M32, MK-7684, AB- 154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (suchas, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).D. Examples

[0127] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.1. Example 1(1) Characterization of recombinant AAVs as gene therapy vectors in vitro.

[0128] Six AAV-based drug products (Glybera, Luxtuma, Zolgensma, Upstaza, Roctavian, and Hemgenix) have been approved by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA). Three AAV vectors for cancer therapy (recombinant AAV2-CEA and AAV2-MUC1) are in phase Eli clinical trials. The HSV-1 TK is also being tested for suicide gene therapy in clinical trials. This Aim will characterize two AAV constructs that were modified to be more specific to KSHV-associated cancers. Differences will be determined in amount of therapeutic gene expression and cell killing efficacies with / without SAHA. The pair of KSHV-infected and non-infected cancer cells will be used to evaluate enhanced specificity for cell killing for the KSHV-infected cells.

[0129] The vector encodes two copies of TR, sub-cloned from our pBS-TR vector, and ligated Ori-RNA promoter downstream of the TR fragments (Fig. 10A). mCardinal cDNA are cloned and monitored differences and duration of mCardinal expression changed by the KSHV infection (Fig. 10C, 10D, and 10E). Increased target gene expression was examined with TR fragments in the vector by measuring mCardinal expression (Fig. 10E). mCardinal is later replaced with TP53, and p53 expression was also confirmed with RT-qPCR. For the study, two poly(A) sites were compared, one originated from reporter vector (pGL3 -basic), the other one is from AAV therapeutic vector. Having WSPR motif increases mRNA stability approximately 10- fold (Fig. 10G). Thus, WSPR-containing poly(A) signal was selected (Fig. 10A). To distinguish with endogenous TP53, our exogenous TP53 is codon-optimized and exogenous p53 transcriptswere monitored separately from endogenous p53. Similarly, HSV-1 TK was cloned as a therapeutic gene for the indirect gene therapy. Addition of the HSV-1 TK is to establish an approach, which is independent of the status of p53 mutation.

[0130] Characterizing the biological activity, therapeutic gene expression, and in vitro cancer cell killing efficacies with / without combination with SAHA. Both KSHV-infected and noninfected cancer cell lines will be included to evaluate the KSHV etiology as a cell killing advantage. This will be examined with (i) transcription analyses (RT- qPCR, RNA-seq) to determine robustness of therapeutic gene expression, (ii) cell killing studies (MTT assays), and (iii) protein expression (immunofluorescence assays and immunoblotting). In the end, endothelial colony forming cells (ECFC) will be prepared from iPSCs (Fig. 11). The most recent studies suggested that ECFC is likely an origin of Kaposi’s sarcoma, and the cells were used to confirm the results. a) Transcription studies.

[0131] The recombinant A A Vs expressing TK or mCardinal will be infected with multiplicity of infection (MOI) 103, 104, or 105 with / without SAHA, and total RNAs will be harvested at 48 hours transduction. The SAHA has been evaluated in a clinical trial for KSHV- associated malignancies and the drug is known to robustly reactivate KSHV from the infected tumor cells. The AAVs were purified by a commercial source, and they also determined viral titers with quantitative-PCR. To select the cell model that can be effectively infected with AAV8 and used for KSHV studies, several adhesion cell lines were screened with mCardinal expression. The results showed that SLK cells (renal carcinoma cells line) and 293 cells can be infected with AAV8. KSHV naturally infected primary effusion lymphoma cells (B-cell lines; BC1, BC3, BCBL-1) were largely refractory for AAV8 infection without additional engineering for capsid sequence. Accordingly, SLK cells and 293 cells will be used with / without KSHV infection in this aim. The AAV8-TR2-Orip-TK (AAV-TK) or AAV8-TR2-Orip-TP53 (AAV- TP53) will be used for this study, and AAV-TR2-Orip-mCardinal (AAV-mCardinal) will be served as control. At post 24 hours infection, SAHA will be added to the culture media and examine enhancement of transgene expression. RNA-sequence analyses will also be included. The results will be analyzed by DEseq2 using the Nextflow pipeline to comprehensively extract differentially expressed (DE) genes along with their q-values. The extracted DE genes (fold change >2 and q<0.05) will be utilized for the enrichment analyses, GSEA and DAVID to identify newly activated or inactivated pathways. The cellular responses by the AAV infectionshould be comprehensively identified by DE genes with the most significant expression changes in newly activated pathways. Such transcripts may also provide additional target to further enhance the cancer cell killing. For example, if anti-apoptotic gene expressions are identified or certain metabolic pathways are activated by the gene therapy, they will be inhibited either pharmacologically or genetically. The TP53KO cells will be generated with KSHV-infected SLK cells, and compare the transcription profiles.

[0132] Transcription studies on KSHV genes: Because the mechanisms of HSV-1TK / GCV is inhibition of DNA replication via toxic GC V-triphosphate, and the GCV is an anti-herpesvirus drug, the inhibition of the KSHV replication swill be evaluated by measuring late gene transcription and genomic copies with qPCR. GCV will be incubated at 24 hours post AAV infection. Without GCV or no AAV-infected cells will be used as negative controls. KSHV reactivation will also be stimulated with SAHA and efficacies of having exogenous (additional) HSV-1 TK will be examined. An oncolytic approach, in which endogenous KSHV is reactivated by bortezomib and SAHA to enhance cancer cell killing has shown promising results, and safety and oncolytic effects with bortezomib is being evaluated in clinical trial at the NCI. That combination with our AAV with GCV should further facilitate the cancer cell killing in addition to prevent KSHV from replication. Comparing with / without AAV-TK and with / without SAHA revealed if AAV-TK can be an adjuvant for the oncolytic approach. b) Cell killing studies (MTT assays).

[0133] Effects on cell viability by induction of p53, HSV-1 TK / GCV, or mCardinal (control) will be determined after 48 hours of rAAV infection. The same sets of cells will be utilized described above and effects of KSHV-infection and the status of the p53 mutation will be revealed. The TK construct will be incubated with GCV at 24 hours post infection and continued to incubate another 24 hours. Bystander effects will be examined by reducing amount of AAV infection (e.g. MOI 103) and examine surrounding (non-transduced) cell killing effects. The HSV-1 TK will be stained with specific antibody and examine if the no-TK expressing (transduced) cells are also killed in the presence of GCV by Flow cytometry analyses with dead / live staining. Non-infected cells and rAAV-mCardinal infection will be used as negative controls.

[0134] For MTT studies, three different MOI (103, 104, 105) will be used initially and efficacies of cell killing by measuring IC50 value will be determined. Flow cytometer analyses with dead / live staining will be accompanied. SAHA will also be included to determine if it cansynergize the cell killing, if so how effectively. KSHV-infected cells should be killed more frequently than non-infected cell group. SAHA should have more synergistic cell killing effects in KSHV-infected cells due to induction of K-Rta expression. The synergistic effects will be calculated with SynergyFinder 3.0. c) Target protein expression and flow cytometry analyses.

[0135] The protein expression and cell apoptosis and / or autophagy induction will be measured by staining with AnnexinV or transduction of EGFP-LC3 by Flow cytometry. Immunoblotting will further confirm the exogenous protein expression (p53 and HSV-1 TK) and also examine cellular proteins to monitor cell apoptosis, cell cycle arrests (cleaved PARP1, p21, Caspase), and / or autophagy inductions. This is done because transcription does not necessarily associate with protein amount and protein actions. Many protein markers associated with specific cell death pathways are also known. Combination of transcription profiles with protein analyses bolsters the evidence for the cell death mechanism.

[0136] Results: 1. The overexpression of p53 alone strongly induces cell apoptosis and synergistic effects or additional advantages of having KSHV infection become difficult to analyze. In that case, AAV will be further diluted and the minimum MOI required for IC will be identified. 2. SAHA does not synergize or SAHA itself has dominant effects on cancer cell killing. In that case, other cancer drugs will be utilized that are known to induce KSHV reactivations. These drugs include BET inhibitor (OTX015) and bortezomib. Small molecule drug libraries are commercially available. The most of cancer therapies performed clinically are combination therapies to reduces toxicity (amount) of individual drugs and increases efficacies of cancer cell killing. Administrating AAV directly to KS lesions in the combination with small molecular drugs is expected to be the most reasonable approach to target heterogenic KSHV- associated cancers.(2) Characterization of recombinant AAVs as gene therapy vectors in xenograft model.

[0137] AAV therapeutics is administrated to mice to evaluate tumor growth inhibition. Xenograft studies are performed with KSHV-infected primary effusion lymphoma cells. The sub-cutaneous injections containing BCBL-1 (p53 mutation) and BC-3 (p53 wild type) with / without SAHA are injected, after mixing with matrigels. Second, the effects of the KSHV infection is examined. KSHV-infected GM12878 cells (an EBV-infected lymphoblastic cell line: LCL) is utilized and compared to the therapeutic effects with parental GM12878 cells. TheGM12878 cells are infected with KSHV in vitro. The toxicity is measured by blood chemistry and immunohistochemistry. KSHV-infected SLK cells and ECFC will be used for xenograft studies. The sub-cutaneous (SQ) injection model will be performed by using Matrigel with / without SAHA. The SQ model allows us to inject AAV locally. The toxicity is determined by blood chemistry and immunohistochemistry at the end of xenograft studies. a) The SQ injection model evaluates tumor growth inhibition with a direct targeting approach.LO 138 J Five million SLK2. 19 or ECFCr2.19 cells stably expressing luciferase will be either treated with mCardinal-AAV / AAV- p53 at MOI 104 or untreated in vitro for four hours (Fig. 11). The cells will be collected, washed twice with culture media, mixed with an equal volume of Matrigel (200 pL), and subcutaneously injected into the flanks of 6-to-8- week-old male and female NRG mice (8 mice per group). The tumor growth (size) will be monitored every other day, and luminescence, which indicates number of luciferase-expressing cells, will be measured every 8 days. Secondly, synergistic effects with SAHA will be determined by including a group with SAHA treatment. The SAHA will be administrated in drinking water (1.4g / L) and compared among AAV-mCardinal with / without SAHA and AAV-p53 with / without SAHA groups. When the largest tumors reach up to 2 cm or at day 32, mice will be sacrificed, and the tumors and major organs will be collected at the time. The size and weight of the tumors will be determined and fixed with a formaldehyde solution for IHC analysis and TUNEL staining, or frozen in liquid nitrogen for later analyses with immunoblotting and RNA-sequencing. For IHC, KSHV LANA, p21, Ki67, and p53 will be stained to monitor effects on xenograft tumor cell proliferation. In addition, blood is drawn, and serum is isolated at the end of the experiment to examine organ damage caused by the treatments. b) The SQ injection model to evaluate tumor growth inhibition with an indirect targeting approach.

[0139] For AAV-TK construct, SLKr2.19 or ECFCr2.19 cells will be used that are stably expressing luciferase and will be treated with AAV-TK at MOI 104 in vitro (Fig. 12). AAV- mCardinal treated BCBL-1 will be used as a control. The AAV-TK or AAV-mCardinal treated cells will be similarly injected by mixing with Matrigel, and subcutaneously injected into the flanks. Once tumors reach 1 cm, mice will be randomly separated into 2 groups, and one of the groups will receive GCV (50 mg / kg) by i.p. once a day, and the other group will receive PBS.The tumor growth (size) will be monitored every other day, and luciferase activity will be measured every 8 days that will examine tumor metastasis with in vivo luminescence imaging. Secondly, synergistic effects with SAHA is determined by including a group with SAHA treatment as described above. AAV- mCardinal + GCV with / without SAHA and AAV- TK+GCV with / without SAHA groups will be compared. When the largest tumors reach up to 2 cm or at day 32, mice will be sacrificed, and the tumors and major organs will be collected at the time. The size and weight of the tumors will be determined and fixed with a formaldehyde solution for 1HC analysis and TUNEL staining, or frozen in liquid nitrogen for later analyses with immunoblotting and RNA- sequencing. For IHC, KSHV LANA, p21, Ki67, and HSV-TK will be stained. c) Studying specificity to the KSHV etiology in xenograft model.

[0140] How much of having two copies of TR with KSHV K-Rta responsive promoter makes the vector specific to KSHV infected cells will be examined. Here, KSHV-SLK and parental SLK will be utilized for xenograft studies. While it is understood SLK cells do not represent KS tumors very well, unfortunately there are no good xenograft model available. Nonetheless, SLK cells have been used widely to examine efficacies of drugs or significance of specific gene in cancer cell growth in mouse.

[0141] For GM12878 xenotransplantation, randomized NRG mice (n = 8 mice / group) will be inoculated with 5 x 106 KSHV-GM12878 or parental GM12878 cells in 100 pL PBS intravenously (IV) into the lateral tail vein of mice (Fig. 13). The GM12878 cells will transduce luciferase cassette and generate luciferase stably expressing GM12878 cells. This step helps to perform in vivo live imaging to track tumor growth. Two days after GM12878 injections, AAV- mCardinal or AAV-TK will be administrated via IV. Based on previous experiences with AAV- vIL- 6 mouse studies, 2 x 1013 / kg AAV-TK will initially be injected. The suitable amount of the AAV will be confirmed by the presence of HSV-1 TK expression in isolated GM12878 cells from mice blood. Flow cytometry will be used to determine after co-staining HSV-1 TK and luciferase. Toxicity in mice after GCV administration will be monitored. The minimum amount of AAV HSV-1 TK will be determined, which infects xenografted GM12878 more than 80%. If AAV TK infection to GM12878 is limited, AAV HSV-1 TK will be administrated repeatedly at the time of imaging by combining with luciferin (weekly). Two days after the initial AAV injection, GCV will be administered daily (50 mg / kg i.p.). GM12878 cell growth will be tracked by measuring luminescence. The luminescence intensity after injection of luciferin will bemeasured weekly for three weeks. By comparing between KSHV-infected and non-infected as well as GCV-treated or non-treated, efficacies of AAV TK will be revealed as a specific suicide gene therapy for KSHV-associated tumors.

[0142] Results: The xenograft studies will evaluate the efficacies of tumor cell killing vs. toxicity of the gene therapy vector to the mouse. First, the SQ model should largely avoid toxicity of AAV infection to mouse normal tissues, because excess AAV will be washed away before tumor injection. The advantage of this approach is to target cancer cells more accurately with specific amounts of AAV. The amount of AAVs infected to the target cells by qPCR will be confirmed before implanting tumor cells in mice. Because p53 expressing adenovirus vector has been used in the human clinics, low toxicity to mouse tissues (normal cells) by p53 overexpression is to be expected. The toxicity should be further reduced with the use of a KSHV specific promoter for the vector (Fig. 10). However, there would be general toxicity with HSV- TK / GCV with the systemic injection approach (IV). This is because the AAV will infect most mouse tissues especially in the liver. Accordingly, liver damage with serum chemistry is monitored. If the toxicity is found to be significant, the SQ model similar to SQ injection model will be performed with a pair of GM12878 cells. The amount (1.4 g / L) of SAHA in the drinking water was reported not to induce weight loss for 3 weeks in a previous publication. Depending on the progress, the physiological properties (homogeneity) of the purified AAV are determined by tomography.2. Example 2

[0143] The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes with arrays of transcription factor binding sites. Here, it has been shown that KSHV TR possesses a transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies to TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27 Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K- Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.

[0144] Enhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatial and quantitative expression. Here, it is shown that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHVinducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat(TR) fragments to lytic gene promoters drastically enhanced KSHV replication, transcription activator, and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

[0145] There is growing awareness of the need to understand the spatial and temporal organization of the genome structure and its role in regulating gene expression. Given that nuclear enzymes and transcriptional factors are in limited supply and cannot be everywhere in the nucleus at the same time, the nuclear architecture (including chromatin structure and protein distribution) must allow for these molecules to be concentrated at the proper time and place for coordinated gene expression to occur. Understanding the three-dimensional (3D) structure of the genome and its impact on neighboring genomic elements is, therefore, critical to understanding gene regulation. The significance is also highlighted by the fact that the deregulation of genomic interactions by mutations of intragenic genomic regions and nuclear remodeling factors such as SWItch / Sucrose Non-Fermentable (S WI / SNF) frequently leads to diseases. Assisted by the three-dimensional genomic architecture, gene enhancers are known to act in cis and an orientation-independent fashion by forming genomic loops to increase frequencies for transcription initiation at promoters. By forming genomic looping with multiple promoters, the enhancer DNA fragments neighbors and activates many promoters at the same time. A largergenomic region collectively bound by an array of transcription factors at higher density, hence harboring a higher density of transcription enzymes, is called a super-enhancer. Cellular proteins, such as mediator complex subunit 1 (MED1) and bromodomain-containing protein 4 (BRD4), are known to be located at the super-enhancer region, and those proteins further compartmentalize nuclear condensates with their intrinsically disordered domain for maintaining selective gene expression, hence cell identity. The genomic interaction between enhancer and promoter mediated by transcription-related enzymes positions the RNA polymerase II (RNAPII) pre-initiation complex at specific genomic sites for sensitizing inducible gene expression with specific signaling events that activate enhancers.

[0146] The Kaposi's sarcoma-associated herpesvirus (KSHV) viral genome consists of an approximately 140-kb unique coding region flanked by multiple terminal repeats (TRs)of 801 bp with high GC content. KSHV genomes persist in latently infected cells as episomes via tethering to the host cell chromosomes. During latency, the expression of lytic viral genes is poised to be transcribed, and only a few latent genes are actively expressed and translated. Among these latent genes, ORF73 encodes latency-associated nuclear antigen (LANA), which plays a crucial role in latent episomal DNA replication and segregation during host cell mitosis. The TR contains a DNA replication origin celleporid-P that consists of two LANA-binding sites (LBSs): a higher affinity site (LBS1) and a lower affinity site (LBS2) followed by an adjacent 32-bp GC- rich segment. Episome maintenance requires at least two LBS 1 / 2 binding sites, and the viral genome consists of 30-40 TRs. A crystal structure demonstrated that the LANA DNA-binding domain (LANADBD) mainly exists as a dimer in solution. Five LANADBD dimers can interact end-to-end to form a decametric ring with an exterior diameter of 110 A and an interior diameter of 50 A, and the inner diameter is sufficiently large to accommodate double stranded DNA. DNA binding further induces oligomerization of LANADBD, and hydrophobic interface between LANA dimer to form the decametric ring is essential for cooperative DNA binding and DNA replication, hence episome maintenance. The specific LANA’s TR DNA binding also increases up to 600-800 LANA copies [2 (dimer) x 5(decametric ring) x 2 (two binding sites per TR unit) x 30-40 (TR copies per episome)] to locate a single KSHV episome. As a result, LANA can be seen as LANA dots in KSHV-infected cells with immunostaining. Because KSHV latent chromatin is circular, the TRs always localize relatively close to the unique region that encodes all inducible genes. KSHV replication and transcription activator (K-Rta) protein are known to activate those inducible promoters. The isolated reporter constructs showed that K-Rta can activate at least 33 lytic gene promoters in 293 cells. Previous studies indicated that TR possesses transcription regulatory functions in reporter assays. In this report, it is shown that the TR is (i) encoding an array of transcription factor(LANA) -binding sites, (ii) recruited by transcription-related enzymes including BRD4, (iii)modified by a histone H3K27Ac mark, (iv) expressing nascent RNAs, and (v) possessing an orientation-independent transcription activation function. A model has been proposed in which KSHV TR is a large transcription regulatory domain (enhancer) for KSHV inducible gene promoters (unique region).3. Example 3 a) AAV8-TR2-Orip-TK with Ganciclovir (GCV) induced cancer cell death in a KSHV infection-specific manner.

[0147] An AAV therapeutic expressing thymidine kinase was prepared. In gene therapy, the thymidine kinase (TK) expression is regulated by KSHV's enhancer and promoter. Therefore, KSHV-infected cell death (but not non-KSHV-infected cells) was induced. To assess the cell line bias, we established two KSHV-infected cell lines (293 cells and iSLK cells) and used parental non-infected cells as comparisons. KSHV-infected (+) and non-infected (-) cells were first transduced with AAV8-TR-Orip-TK. Twenty-four hours post-AAV-TK transduction, we incubated with GCV (5pM) with or without OTX015 (BRD4 inhibitor). Previous reports showed that the BRD4 inhibitor induces KSHV reactivation, expecting increased TK expression from AAV therapeutic vectors. The results showed that transduction of AAV-TK induced cell death only in the presence of GCV in both iSLK cells and 293 cells (Fig. 18A and 18B). Most importantly, the presence of latently infected KSHV strongly sensitized the AAV-TK therapy (pink shadows). In the case of 293 cells, KSHV infection is essential to inhibit cell growth by the AAV-TK; the results suggest that the TR-Orip activation is strictly regulated by KSHV infection. iSLK cells, which exogenously encode KSHV transactivator (K-Rta) inducible cassette, may weakly activate TK expression from the AAV vector via leaky K-Rta expression. Fig. 18C shows the morphology of the iSLK cell on Day 2. b) Bystander effects with AAV-TK with GCV.

[0148] To assess the bystander effects with AAV-TK+GCV therapy, recombinant KSHV were generated, which expresses Histone H2B-mCherry fusion protein. The EFla-EGFP cassette were replaced in the BAC backbone with EFla-H2B-mCherry. AAV-TK was transduced to EGFP(-i-) KSHV-infected iSLK cells, and EGFP(+) cells were mixed with H2B- mCherry(+) KSHV-infected iSLK cells. Combined iSLK cells (Red and green) were thenincubated with GCV. The bystander effects were assessed with the viability of H2B-mCherry (+) cells. As shown in Fig. 17E, H2B-mCherry-positive cells were also killed in the presence of GCV in an AAV-TK transduction-dependent manner. These results suggest that AAV-TK transduction does not need to be 100% of KSHV-infected cells to eradicate the cancer cells. c) RESULTS(1) Latent KSHV chromatin modification maps

[0149] To understand the KSHV latency-lytic switch, a comprehensive histone modification of the KSHV episomes in naturally infected cells was revisited. A public database and our cleavage under targets and release using nuclease (CUT&RUN)data sets were used. If having a specific regulatory genomic domain is necessary for maintaining inducible KSHV latent chromatins, cell lines that can produce infection virions with stimulation should conserve similar genomic domains in KSHV latent chromatin. Four histone modifications primarily associated with active transcription (H3K4mel, H3K4me2, H3K4me3, and H3K27Ac) and repressive marks (H3K27me3, H3K9me3) were examined. While attempting to locate H3K9me3, reasonable peaks with two independent antibodies could not be identified. Figure 1A depicts histone modifications of KSHV latent chromatin in BCBL-1 cells. The count per million (CPM) normalized peaks were mapped on KSHV genomes. The results showed that four active histone marks are clustered mainly at genomic loci encoding early and immediate-early genes, which is consistent with previous reports. The H3K27Acmarks were restricted at two Ori-Lyt regions, PAN promoter, viral interferon regulatory coding loci, LANA promoter, ORF75 promoter, and K15 promoter regions (Fig. 1A). Although H3K4mel and H3K4me2 marks are colocalized with H3K4me3 marks, H3K4meland H3K4me2 modifications were more broadly distributed around the H3K4me3 marks. The H3K4me3 modification showed sharper peaks than H3K4mel and me2 marks inBCBL-1 (Fig. 1A). While it is difficult to compare between peaks at the unique genomic region of the KSHV genome (one copy per genome) and that of TR fragments (30- 40copies per genome), there are still noticeably strong H3K4me3 and H3K27Ac signals(more than 10 times) at the TR regions but not with the H3K4mel, 2, or H3K27me3 (Fig.lA, right panel).

[0150] On the other hand, repressive marks (H3K27me3) were localized more broadly in BCBL-1. Other KSHV naturally infected primary effusion lymphoma cells or experimentally infected iSLK cells showed similar histone modification occupancies for active histone marks, especially for H3K4me3 and H3K27Ac marks. However, H3K27me3 had different degrees ofsignals at late gene cluster regions. With that, BC3 cells showed more H3K27me3 modification at late gene cluster regions, while BC1 cells showed little signals at the same regions (Fig. IB). The experimentally infected iSLK cells also showed patterns more resembling BC3 and largely occupied by H3K27me3-modified histones. These results indicate that maintaining active genomic regions is a more conserved trait than the repressive mark for KSHV latent chromatin.(2) TR region is actively transcribed in latently infected cells

[0151] Significant modifications by the active histone marks (H3K27Ac and H3K4me3) suggested that TR possesses an enhancer function. To assess if TR is an active enhancer, the transcriptional activity at TR was examined next. Nascent RNA sequencing, which measures enhancer RNAs, provides more direct evidence of enhancer activity.The previous GRO- sequence data was reanalyzed to identify nascent transcribing RNAs in the TR region, and nascent RNAs were mapped on the KSHV genome. The results showed that TR is actively transcribed during latent infection, and reactivation slightly reduced the amount of nascent RNAs from TR (Fig. 2).(3) Protein interaction among K-Rta and LANA-interacting

[0152] The latency-lytic switch mechanism includes protein-protein association networks of KSHV LANA and K-Rta-interacting transcription-related enzymes using the STRING database. In this analysis, functional interactions between two proteins were visualized by the number of lines among protein nodes. The line between two nodes indicates either experimentally validated interaction, coprecipitated proteins in the proteomics database, or predicted interaction based on coexpression and functionality. An increased number of lines between two nodes usually suggests higher protein-protein interaction confidence.

[0153] KSHV LANA is essential for establishing and maintaining latent infection, and proteins localized in proximity to LANA were previously identified. Among the proteins localized in proximity to LANA (Fig. 3A), cellular CHD4 protein plays a key role in establishing and maintaining latent infection. Proximity-biotin labeling with recombinant KSHV also confirmed that LANA interacts with BRD4 and also identified components of the Imitation SWItch complex and several others as LANA-interacting proteins [Fig. 3, blue oval]. In addition, to reveal how K-Rta triggers KSHV reactivation, cellular proteins that are induced to interact with RNAPII were also reported in the presence of K-Rta during KSHV reactivation. In the latter study, rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) was applied. The method is suitable for identifying transcription factor complexes on chromatin.By identifying proteins that interact with RNAPII and K-Rta during reactivation on chromatin, critical proteins for K-Rta trans activation were isolated. The study identified that K-Rta interacted with NCoA2, SWI / SNF, and mediator complex, and these proteins form a large protein complex (Fig. 3B). The results were consistent with previous reports that showed K-Rta interaction with NCoA2 and SWI / SNF and mediators. To provide insights into dynamic protein interactions between two proteins during reactivation, putative protein interactions among two proteins (Fig. 3C) were combined and visualized. The result showed that LANA-interacting proteins (blue ovals) interact with components of the K-Rta complex (red ovals) when they are recruited in proximity during reactivation. Nearly all, except two (SMCHD1 and HP1BP3) LANA-interacting proteins were predicted to interact with at least one of the components of the K-Rta complex (Fig. 3A). The protein interaction model suggests thatcomponents of the LANA protein complex at TR (during latency) can be flexible whenK-Rta brought its protein complex near the LANA binding sites during reactivation.(4) Determining LANA-interacting protein recruitment sites on the KSHVgenome

[0154] To gain insights into the gene regulation, occupancies of the LANA-interacting proteins by CUT&RUN in BCBL- 1 (Fig. 4A) and BC-1 (Fig. 14) were examined next. Proteins were selected based on the availability of antibodies that have been proven for successful CUT&RUNs and immunofluorescence studies. The results showed thatLANA-interacting proteins and enzymes (BRD4, ADNP, KMT2D, SMARCA5, and CHD4)are all colocalized with KSHV LANA on latent chromatin (Fig. 4A). TheLANA-interacting proteins were highly enriched at the TR region when compared with occupancies at the unique region (Table 1). The LANA sequence reads TR were significantly enriched at TR (Table 1). No comparable signals were seen with any other proteins tested or genomic regions. With the criteria based on relative enrichment at TR, at least RNAPII, LANA, BRD4, and CHD4 localize at TR in latently infected cells. The lack of specific enriched peaks for transcription factor binding for the CTCF or SMC1 at TR also suggested no cohesin-mediated fixed genomic loops with TR. Noteworthy, occupancies of those enzymes at TR were not equal, even though those transcription factors were found to be in close proximity to LANA (Fig. 3A). The results indicated either highly heterogenic recruitment at an individual episomal level in an infected cell or dynamic protein recruitment at the individual TR copy within an episome. If the interacting proteins were recruited equally by LANA interactions, there would be peaks similar to LANA (Table 1).(5) Nucleosome organization at the origin of lytic DNA replication

[0155] The observation, in which LANA-interacting proteins (transcription factor complex for latent maintenance) occupied epigenetically active unique regions with poised RNAPIIand the latent chromatins were not actively transcribed, suggested that pausing transcription initiation at the genomic region plays an important role for latency. Zooming the KSHV genomic region (22,000-30,000), encompassing Ori-Lyt and PAN RNAcoding regions, where the KSHV latent chromatin possessed conserved active histone modifications among infected cells (Fig. IB), showed that LANA, CHD4, ADNP, SMARCA5, and BRD4 are recruited next to the CTCF / SMC1 peak (Fig. 4B). The CTCF / SMC1 binding site also creates a small boundary of the transcription regulatory domain at the site(Fig. 4B, CTCF binding sites marked with green shadow). The LANA complex occupied the H3K27Ac nucleosome localized next to the RNAPII peak (Fig. 4B, red shadow), suggesting that the H3K27Ac-nucleosome plays a critical role in stalling the RNAPII. TwoK-Rta binding sites were clearly positioned next to the LANA-bound nucleosome. Directionality and position of CTCF / SMC1 as well as previous Hi-C sequence, suggested that Ori-Lyt forms small genomic loops with the PAN RNA promoter region, and the genomic loop harbors the paused RNAPII (a combined figure with previous Hi-C studies is presented in Fig. 15). The RNAPII peaks at Ori-Lyt are the strongest, when the TR copy number in all three PEL cell lines (Fig. 4B) was considered. The results suggested that the majority ofinfected episomes in a cell in PEL cell lines harbor the pausedRNAPII at the same position (Fig. 4B; Fig. 14). Because K-Rta binding sites were positioned next to the LANA-bound H3K27 Ac- modified nucleosome and the K-Rta protein complex contains SWI / SNF (Fig. 3), K-Rta was examined to see if expression evicts the nucleosome and triggers transcription elongation. Indeed, the nucleosome occupancies were significantly decreased when the reactivation was triggered with K-Rta expression(Fig. 4C). Furthermore, the GRO-sequence showed strong induction of transcription elongation at the genomic region during reactivation (Fig. 2). Based on the conserved histone modifications (Fig. 1) and protein recruitments (Fig. 4; Fig. 14), local 3D genomic structure with the position of poised RNAPII (Fig. 15), and robust nascent RNA expression with reactivation stimuli (Fig. 2), it is suggested that a KSHV latency-lytic switch is triggered by the initiation of transcription elongation at the Ori-Lyt through the recruitment of theK-Rta complex (Fig. 4D).(6) Genomic interaction between TR and unique region

[0156] Having similar protein complexes recruited at selected genomic loci in the unique region and TR, it was determined that these two genomic regions were located closer to the 3D genomic structure. To study this, previous Hi-Csequence data sets were reanalyzed with TREx- K-Rta BCBL-1 cells. KSHV TR fragments were first isolated, and the position of ligated KSHV DNA fragments was mapped onto the unique region of the KSHV genome. The results showed that a significant majority of TR fragments(>99.5%) were ligated with other TR fragments (Fig. 5A), suggesting that individual TRunits are highly compacted with each other. The result agreed with studies on LANANBs with super-resolution fluorescence microscope analyses and indicated that KSHV TR fragments formed a self-aggregate with LANA and LANA-interacting proteins. Even though overall frequencies are much less (<0.5% of total TR genomic loops), two genomic regions formed genomic loops more frequently with TR (Fig. 5A). These regions were within the Ori-PAN RNA transcription regulatory domain (Fig. 15) and near the K-Rtapromoter region. The induction of KSHV reactivation by K-Rta expression increased the overall KSHV- KSHV genomic loops, which also increased the genomic interaction with TRs (Fig. 5B). To further quantitatively assess the interaction between TR and the unique region in latent and reactivated cells, the normalized read counts for each condition were calculated and then evaluated the ratio of read counts for the unique regions to TR regions. The ratio increased in lytic-infected cells, suggesting an increasing interaction between TR and the unique region during reactivation (Fig. 5C). Considering CTCF to be frequently localized at epigeneticallyactive genomic regions, having no CTCF binding sites within 25 kb+ of highly active DNA fragments (Table l)would be a unique genomic feature. This design should make the TR a mobile enhancer for KSHV inducible genes because the TR fragments are maintained to be in proximity to the inducible viral promoters with the episomal genomic structure. In summary, TR is a flexible large protein storage containing LANA and its interacting proteins such as CHD4 and BRD4. The protein / TR aggregates were then maintained in proximity to inducible promoters, especially to the Ori-PAN RNA and K-R promoter regions in 3D (Fig. 5D).(7) TR supports LANA and K-Rta transcription function

[0157] The H3K27Ac histone modification and enrichment of transcription enzymes suggest that TR possesses a transcription regulatory function when it localizes proximity to a promoter. To study this directly, TR from KSHV BAC16 was first cloned by homologous recombination with recombineering technique (Fig. 6A). Homology arms are included in the long primer sequence (Table 2), and the primers were used to amplify the pBlueScript plasmid vector. The resulting PCR fragment containing plasmid DNA'sorigin, ampicillin-resistant cassette, and partial multiple cloning sites was transduced into BAC16 containing Escherichia coli. The successful recombination generates circular plasmids in BAC containing E. coli, which outgrowths via high copy origins of plasmid DNA replication. The ampicillin-resistant colonies were then screened for the presence of TR, and the number of TR copies was examined by the size of plasmid DNA after restriction enzyme digestion (Fig. 6B). To study the association of increased TR copy numbers with transcription function, the pBlueScript- TR (pBS-TR) vector encoding 0, 2, 4, and 6 copies of TRs were isolated. The luciferase reporter cassette was cloned into the pBS-TR vectors. KSHV reporter libraries generated with the pGL3 basic vector were used as a PCR template. The reporter cassette includes synthetic poly(A) sites upstream of the inserted promoter and SV40 poly(A) sites downstream of the luciferase gene (Fig. 6C). Reporter constructs were then cloned in two orientations (left and right: Fig. 6C)and examined effects on directionality. The results demonstrated that the basal KSHVPAN RNA promoter activity was increased independent of the orientation in 293FT cells in the presence of the TR fragment. The results suggested that the TR region has an enhancer activity since enhancers are known to act independently of the orientation (Fig. 6D). However, the TR-left PAN promoter, in which TR fragments are physically closer to the PAN RNA promoter, showed higher luciferase activity. Including the TRfragment in the reporter also synergized K-Rta-mediated reporter activation (Fig. 6E). On the other hand, while the presence of TR increased basal promoter activity (TROvsTR4 with vector control in Fig. 6G), LANA acquired a stronger gene repression function only in the presence of TR. The effects were exaggerated when the LANAIDR domain (Fig. 6G) was deleted. The results suggested that TR fragments increased the basal levels of transcription activity (TRO vs TR4) and enhanced the K-Rta transactivation functions synergistically. To study the association between TR copy number and transcriptionactivity, the reporter was cotransfected with TR2, 4, and 6 with K-Rta. The results showed that increasing the TR copy number enhanced promoter activity; however, the effects were not always linear in 293FT cells. The results showed that the enhancement of the promoter activity was saturated with four copies of TR in the reporter assay (Fig. 16). Finally, the enhancer activity was tested with KSHV- infected 293 FT cells. Infectious KSHV was first prepared from BAC16-Wt iSLK cells and generated KSHV-infected293FT cells with hygromycin selection (Fig. 7A). The KSHV infection was confirmed with immunofluorescence staining and immunoblotting (Fig. 7A and B). The KSHV-infected 293FT cells were then co-transfected with K-Rta expression plasmid with reporter constructs and examined enhancer activity. The results again showed that TR enhanced K-Rta-mediated transcription function synergistically, even more so than in non- KSHVinfected 293FT cells (Fig. 7C). The transfection efficacies were determined by the proportion of the RFP -positive cell population and showed that there were no obvious differences in transfection efficiency (Fig. 7D).

[0158] Taken together, it is proposed that KSHV TR is an enhancer for both transcription activation and repression with K-Rta and LANA, respectively. Our study suggests that a very clever design of the KSHV enhancer, which encodes arrays of LANADNA-binding sites to create phase-separate LANA NB at TR, mechanism positions LANA to control KSHV lytic gene promoters during latency. d) DISCUSSION

[0159] While KSHV promoter activity has been measured in isolated reporter plasmids, and a number of key cellular transcription factors were identified as important regulators for the KSHV latency-lytic switch, a mechanism through which many viral promoters are synchronously regulated remains unclear. This study aimed to provide insight into the spatial and temporal KSHV gene regulation by focusing on the transcription function of TR. The TR region takes up to one-fifth of the highly crowded KSHV genome.

[0160] To identify critical regulatory domains of latent chromatin, a histone modification map was first established with CUT&RUN analyses (Fig. 1). Our CUT&RUN showed the enrichment of each transcription factor or histone modification as a relative peak across the KSHV genome. Because CUT&RUN produces a significantly lower background than ChlP-seq and is very sensitive to the specificity of antibodies, the peak height does not always indicate an abundance of the recruited protein at the specific sites. Differences in affinity and specificity among antibodies and also differences in the total amount of the protein in the cell make significant differences in releasing DNA fragments via incubation with protein- A / G-micrococcal nuclease (MNase). Accordingly, our focus on the CUT&RUNs was to comprehensively identify protein recruitment sites on the KSHV genome and compare the position of peaks among naturally infected cells.

[0161] The histone modification predicts which genomic regions are accessible to DNA- binding proteins. The CTCF, SMC1A, and Hi-C data also helped us to identify key genomic regions that formed a higher density of genomic hubs. Among active histone marks, H3K4mel, H3K4me3, and H3K27Ac are known to be present as enhancers. Subsequent studies showed that H3K4mel is a marker for poised enhancers. When the poised enhancer is activated, the enhancer begins to possess H3K27Ac marks with or without H3K4mel. However, the concept of enhancers has been reconsidered in recent years, and recent models emphasize more for the presence of regulatory elements. With this definition, many regulatory elements with enhancer activity (the ability to enhance distal transcription) can also work as promoters. In contrast, genepromoters, defined by the location near the transcription start sites of the coding sequence, also have enhancer activity. This study described TR as an enhancer because it was shown that the TR region served as a platform to recruit transcription regulatory proteins (Table 1) and as a regulatory domain for KSHV inducible genes (Fig. 6).

[0162] Since latent chromatin is largely silenced, it was initially highly conserved H3K27me3 marks on KSHV latent chromatins. Studies by others elegantly demonstrated the recruitment of the PRC1 / 2 complex on KSHV genomes in iSLK cells. To our surprise, the degree and position of H3K27me3 marks varied among naturally infected three PEL cell lines (Fig. IB) and experimentally infected iSLK cells. Compared with BC3 cells, the amount of H3K27me3 at the late gene cluster in BC1 was much less, and the position of the H3K27me3 also varied among cell lines. Considering that each PEL cell harbors 30+ episomes and studies have been performed with the cell population, only a selected fraction of infected episomes were thought to possess H3K27me3 modifications. Yet, KSHV episomes are not actively transcribed in PEL cells. Accordingly, we suggest that pausing RNAII elongation at the unique region but not global chromatin condensation(s) is the major contributor to silencing inducible gene transcriptions.

[0163] On the other hand, active histone modifications such as H3K27Ac and H3K4me3 were more conserved at specific genomic regions in PEL cell lines and experimentally infected cells. This suggests that maintaining a specific active genomic domain is the strategy that KSHV has evolved to maintain inducible chromatin in the presence of K-Rta. Importantly, these active regions possess the major K-Rta and LANA binding sites, and the K-Rta DNA-binding sites are precisely positioned next to the LANA complex-tethered nucleosome at both Ori-Lyt regions. A high LANA protein concentration at TR can ensure LANA and LANA-interacting protein recruitment at the LANA binding sites in the unique region for latency establishment and maintenance by distantly regulating the inducible promoter. Although more mechanistic studies have to be followed, the recruitment of SWI / SNF via K-Rta DNAbinding evict the nucleosome, hence the LANA complex from the Ori-LytDNA (Fig. 4D). Indeed, an earlier study showed establishing nucleosome-free loci atK-Rta promoter regions during reactivation. It was proposed that the eviction of LANA- tethered nucleosome at Ori-Lyt, where RNAPII paused, is a well- designed and highly efficient KSHV latency-lytic switch mechanism.

[0164] A combination of previous proteomics studies on LANA and K-Rta allowed us to draw the putative protein interaction maps (Fig. 3). It was shown that cellular proteins thatinteract with LANA in latently infected cells can accommodate enzymes that interact with RNAPII and K-Rta during reactivation. Multiple copies of TR recruited a significant amount of LANA and BRD4. Those two proteins have IDR domains and can attract many other histone enzymes as protein hubs. It was speculated that large and highly disordered acidic repeat regions (IDR) of LANA play an important role in capturing such enzymes at TR. KSHV LANA and BRD4 are known to form phase-separate protein condensates that should further facilitate TR fragments to be compartmentalized, which is indeed seen in Hi-C (Fig. 5). The mechanism is analogous to a system operated at cellular super-enhancer with the interaction between two 1DR- containing proteins, BRD4and MED1. Importantly, KSHV K-Rta interacts with MED1 during reactivation (Fig.3), and KSHV LANA associates with BRD4 at TR and Ori-Lyt region (Fig. 4; Table 1). Thus, TR is a well-poised enhancer for KSHV lytic gene promoters, and the recruitment of MED1, a component of the mediator complex, byK-Rta recruitment in the unique region activates viral gene promoters. The recruitment of MED1 creates many inducible genomic loops among BRD4-bound regions [TRs and RNAPII poised regions in the unique region (Fig. 4; Fig. 14)]. Importantly, a genomic looping shift during reactivation was demonstrated, in which the KSHV 3D genomic structure was more compacted by the generation of larger transcription regulatory domains, and 3D genomic structures became squeezed doughnut shapes.

[0165] Suppose the activity of KSHV TR is essential for lytic gene expression. How is theKSHV TR initially activated during de novo infection and maintained to be activated in the infected host cells? Does constitutively active NF-KB and STAT pathways frequently seen in KSHV -infected cells contribute to active episome maintenance? It was speculated that large LANA NBs are like a "solar panel" to extract energy by intercepting cellular signaling events via a highly concentrated LANA / BRD4 complex at TR. The highly concentrated IDRproteins would absorb transcription enzymes from nearing cellular transcription factors because the affinity of interaction among cellular transcription factors and enzymes(i.e., SWI / SNF) is relatively weak in order to share the coactivators among other cellular transcription factors for the dosedependent regulation, except viral transcription factors like K-Rta. The combination of multiple copies of TR fragments and a large unstructured acidic repeat in LANA seems to be designed to concentrate the protein complex near the KSHV's unique protein-coding regions. Consistent with this, a number of transcription factors (e.g., STAT3, p65, IRF4, and BATF) and coactivator enzymes (SMARCA4, SMARCC1) and other proteins that form dynamic and flexible proteincomplexes colocalize with the LANA NBs in BCBL-1. Similar observations have also been reported. It was also shown that inhibition of STAT3 activation at the beginning of KSHV de novo infection attenuated KSHV reactivation. While these ideas await further examination, LANA / TRs can, in return, function like a sponge to awaken the ability of cellular TFs to directly stimulate KSHV reactivation, which should help to maintain the latent state for immune evasion. It was recently shown that the rapid LANA degradation exposed KSHV episomes to the cGAS DNA sensor and consequently triggered KSHV episome degradation.10166 J In summary, KSHV TR was suggested to be an important regulatory domain forKSHVinducible genes by enhancing the transcription function of the KSHV proteins. In contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA NB via a clever genomic design. The regulation of dynamic protein interaction at Ori-Lyt during reactivation and the revelation of how the KSHV episome maintains an epigenetically active state with TR should provide an important target for intercepting KSHV replication cycles. e) MATERIALS AND METHODS(1) Chemicals, reagents, and antibodies

[0167] Dulbecco's modified minimal essential medium (DMEM), fetal bovine serum (FBS), phosphate -buffered saline (PBS), Trypsin-EDTA solution, and lOOx penicillin-streptomycin-L- glutamine solution were purchased from Thermo Fisher (Waltham, MA). Puromycin and G418 solution were obtained from InvivoGen (San Diego, CA). Hygromycin B solution was purchased from Enzo Life Science (Farmingdale, NY). The following antibodies were used for CUT&RUN, immunoblotting, and flow cytometry: rabbit anti-BRD4 (Cell Signaling, E2A7X), rabbit anti-RNAPII (Millipore, clone CTD4H8), rabbit anti-H3K27ac (CST, clone D5E4), rabbit anti-H3K4me3 (Cell Signaling, clone C42D8), mouse anti-P-actin (Santa Cruz, 47778), rabbit anti-gpl30 (CST), and rabbit IgG (CST, clone DA1E).(2) Cell culture

[0168] 293T cells were grown in DMEM containing 10% FBS and lx Pen-Strep-L-Gln at37°C with 5% carbon dioxide air. BAC16-Wt stable iSLK cells were maintained in DMEM containing 10% FBS, 400 pg / mL hygromycin B, 250 pg / mL G418, and lx Pen-Strep-L-Gln at 37°C with air containing 5% carbon dioxide. The KSHV BAC16-Wt infected 293FT cells were established after infection for BAC16-Wt virus with hygromycin selection (1,000 pg / mL) for 2 weeks.(3) Preparation of pTR-reporter construct

[0169] KSHV TR fragments were cloned by adapting the recombineering technique. Homology arms, one targeting the unique region and the other targeting the TR region, were designed and used to amplify the pBlueScript plasmid fragment. The linear PCR fragment that encodes a portion of multiple cloning sites, the origin of plasmid replication, and the ampicillin- resistant cassette was purified from DNA agarose gel. Purified DNA fragments were then introduced to BAC16 containing GS1783 E. coli for red recombination. The ampicillin-resistant colonies were isolated and cultured at 32°C overnight. Outgrowing plasmids were purified with Qiagen Miniprep kits. Purified plasmids were used for restriction enzyme digestions to identify the number of TR copies cloned. The pBluescript plasmid containing TR2, 4, or 6 was then used to transform into stbl3 strain (Invitrogen) for further plasmid amplification. Luciferase reporter fragments were amplified from the pGL3 KSHV promoter library with primers listed in Table 1. The amplified reporter DNA fragment was cloned into pBS-TR multiple cloning sites (EcoRI, PstI, or HincII). The luciferase reporter fragments were cloned for two different orientations. The schematic diagram was also presented in a figure. The expression plasmid for pcDNA HA- K-Rta and pcDNA HA-LANA was described previously. The position of cloned KSHV promoter fragments is listed. For the generation of HA-LANA IDR deletion expression plasmid (pcDNA HA-LANAAIDR), the DNA fragment was synthesized (IDTDNA) and cloned into the pcDNA HA- vector. Synthesized DNA fragments are listed in Table 1.(4) Cleavage under targets and release using nuclease

[0170] CUT&RUN was performed essentially by following the online protocol Dr. Henikoffs lab developed with a few modifications. Cells were washed with PBS and wash buffer [20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine (Sigma, S2626), and proteinase inhibitor (Roche)]. After removing the wash buffer, cells were captured on magnetic concanavalin A beads (Polysciences, Pennsylvania, USA) in the presence of CaC12. Beads / cell complexes were washed three times with digitonin wash buffer (0.02% digitonin, 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and lx proteinase inhibitor), aliquoted, and incubated with specific antibodies (1:50) in 250 pL volume at 4°C overnight. After incubation, the unbound antibody was removed with digitonin wash buffer three times. Beads were then incubated with recombinant protein-A / G-micrococcal nuclease (pAG-MNase), which was purified from E. coli in 250-pL digitonin wash buffer at 0.5 pg / mL final concentration for 1 h at 4°C with rotation. Unbound pAG-MNase was removed by washing withdigitonin wash buffer three times. Pre-chilled digitonin wash buffer containing 2 mM CaC12 (200 LI L) was added to the beads and incubated on ice for 30 min. The pAG-MNase digestion was halted by the addition of 200 pL 2x STOP solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 ug / mL RNase A, and 50 pg / mL glycogen). The beads were incubated with shaking at 37°C for 10 min in a tube shaker at 300 rpm to release digested DNA fragments from the insoluble nuclear chromatin. The supernatant was then collected by removing the magnetic beads. DNA in the supernatant was purified using the NucleoSpin Gel & PCR kit (Takara Bio, Kusatsu, Shiga, Japan). Sequencing libraries were prepared from 3 ng DNA with the Kapa Hyper Prep Kit (Roche) according to the manufacturer's standard protocol. Libraries were multiplex-sequenced (2 x 150 bp, paired-end) on an Illumina NovaSeq6000 system to yield ~15 million mapped reads per sample.

[0171] CUT&RUN sequence reads were processed with fastp and aligned to the human GRCh38 / hg38 and KSHV reference genome (NC_009333.1) with Bowtie2 and yielding mapped reads in BAM files. CPM normalization was applied to show chromatin modification in BCBL-1 in Fig. 1A. Both raw sequenced data and CPM normalized datasets are available under accession number GSE241949. The identical shape of peaks with a spike- in normalization with E. coli DNA genomic DNA from pAG-MNase incubation was also confirmed. Processed files with the spike-in normalization are also available upon request.

[0172] For CUT&RUN quantitative PCR (qPCR) studies, TREx-K-Rta BCBL-1 cells were induced for lytic gene expression by incubat)] to ling with doxycycline (1 pg / mL) and TPA(20 ng / mL) for 48 h. TREx-BCBL-1 cells, induced or non-induced, were used for CUT&RUN, and the amount of released DNA fragments was examined by qPCR with primers listed in Table 1.

[0173] FASTQ files for the Hi-C experiments were processed through the Hi CUP (vO.7.4) pipeline using the KSHV reference genome (NC_009333.1). Bowtie2 (version 2.3.5.1) was used in the pipeline to map valid Hi-C ditags with a single restriction fragment, allowing only unique, high-quality alignments across the genome. Invalid Hi-C ditags, such as dangling ends and PCR duplicates, were removed by the HiCUP Filter script. Valid Hi-C ditags were aligned to the TR region of the KSHV genome with Bowtie2 withal-conc option setting. Each forward and reverse read was independently mapped to theKSHV genome. The extracted paired reads were mapped again to the KSHV reference genome to identify three-dimensional genomic interaction with TR. The mapped reads were visualized with Integrative Genomics Viewer.(5) Reverse transcription- quantitative PCR (RT-qPCR)

[0174] Total RNA was extracted using the Quick- RNA miniprep kit (Zymo Research, Irvine, CA,USA). A total of 1 pg of RNA was incubated with DNase I for 15 min and reverse- transcribed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). The resulting cDNA was used for qPCR. SYBR Green Universal master mix(Bio-Rad) was used for qPCR according to the manufacturer's instructions. Each sample was normalized to 18S ribosomal RNA, and the ddCt fold change method was used to calculate relative quantification. All reactions were run in triplicate. Primer sequences used for RT-qPCR are provided in Table 2.(6) STRING protein interaction visualization

[0175] Names of previously identified (A) LANA neighboring proteins with proximity biotin labeling and K-Rta interacting complex recruited to RNAPII during reactivation were collected and visualized with STRING, a public database of known and predicted proteinprotein interactions. The putative protein interaction among K-Rta and LANA protein complexes during reactivation was also visualized with STRING after manually combining protein names together. The list of proteins based on previous analyses (P < 0.05) with chromatin regulatory function is all included in this analysis.(7) Luciferase assay

[0176] HEK293FT or KSHVr2.19-infected HEK293FT cells were seeded onto 12-well plates at 2.0 x 105 / well. The cells were transfected with an expression plasmid encoding the HA-epitope tagged K-Rta, LANA, or together, along with pBS-TR reporter constructs. The TR reporter constructs encode a varied number of TR repeats and KSHV gene promoter, which is cloned in front of the luciferase coding sequence. Cell lysates were prepared with 1% TritonX- 100 and 0.5% NP-40 in PBS 48 h after transfection. Luciferase activity was measured according to the manufacturer's protocol by using Varioskan LUX (ThermoScientific). At least three independent measurements were performed for each setting.(8) Immunoblotting

[0177] Protein lysates from luciferase assays were subjected to 8% SDS-PAGE gel and transferred to PVDF membranes (Millipore-Sigma, St. Louis, MO, USA). Membranes were incubated with 5% non-fat milk at room temperature for 15 min for blocking and then incubated with the primary antibody at 4°C overnight. After washing the membrane with TBST three times, the membrane was then incubated with horseradish peroxidase-conjugated secondaryantibody (Santa Cruz) at RT for 1 h. The final dilution of the primary antibody was 1:2,000 for the anti-HA tag (Covance) and 1:3,000 for anti-P-actin (Millipore-Sigma) and K-Rta.(9) Statistical analysis

[0178] Experimental replicates of at least three for each sample, including negative controls, were prepared whenever applicable. Results are shown as the mean ± SD from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 9.4.1 software. A value of P < 0.05 was considered statistically significant.4. Example 4: a) Results(1) Preparation and validation of KSHV-inf ection-specific gene expression cassette.

[0179] Recent reports demonstrated that KSHV TR is not only a LANA binding sequence for KSHV episome maintenance but also possesses gene enhancer function. Based on the findings, we devised an idea for a KSHV-tumor-specific gene therapy vector with an AAV. We designed the AAV vector encoding two copies of the TR sequence (Figure 10A), which should help maintain the transduced therapeutic vector in KSHV -infected (LANA expressing) cancer cells and increase KSHV inducible promoter activity. Because the TR enhances KSHV lytic gene promoter activity, we cloned viral lytic gene promoter downstream of the TR sequence (Figure 10B). We selected the Ori RNA promoter (OriP), which is one of the K-Rta direct targets with higher promoter activity, and the genomic fragments possess H3K27Ac and H3K4me3 active histone modifications in infected cells and localize proximity to TR in 3D genomic structure. We utilized an AAV transfer vector as a backbone to generate recombinant AAV for gene delivery. The procedure and vector design are depicted in Figure 10A. As described in more detail below, we cloned fluorescence protein (mCardinal) downstream of the Ori-RNA promoter (pAAV-TR2-OriP-mCardinal). The mCardinal was selected to distinguish the RFP signal produced from the r.219 KSHV viral genome and monitor selective promoter activation in live cells.

[0180] To examine if the assembled enhancer-promoter combination increases the exogenous gene expression in a KSHV infection-specific manner, we transfected the pAAV- TR2-OriP-mCardinal vector into KSHV r.219-infected 293 cells (293 / KSHV cells) or parental 293 cells and monitored mCardinal expression. The signal intensity and the proportion of mCardinal positive cells were measured by flow cytometry. As expected, mCardinal intensitywas approximately 15 times higher in 293 / KSHV cells at 5 days post- transfection (Figures 10C and 10D), and mCardinal signals were also maintained slightly longer in the 293 / KSHV cells (Figure 10E). We also noticed that mCardinal signals were very weak in non-KSHV infected cells, indicating that KSHV-infection, presumably, K-Rta protein expression from infected KSHV genomes, enhanced mCardinal expression. The effects of the TR sequence on enhancing gene expression were further confirmed by transfecting pAAV-OriP-mCardinal vector with or without TR2. Consistent with the previous studies, the vector with TR showed brighter signals in the 293 / KSHV cells (Figure 10F).(2) Preparation of recombinant AAV.

[0181] To conveniently and cost-effectively prepare a large scale of recombinant AAV inhouse, we next adapted the baculovirus-based AAV production platform. With serum-free defined culture media, we could also avoid using animal proteins, which reduces biosafety concerns. AAV therapeutics prepared with recombinant baculovirus platforms are being evaluated in human clinical trials.

[0182] The TR2-OriP-mCardinal fragment was first moved into the baculovirus transfer vector as described in material methods (Figure 10A), and the recombinant baculovirus, which carries the entire AAV transfer genome, including both inverted repeat sequences, was generated. The insect cells were then co-infected with the recombinant baculovirus expressing Rep78, Rep52, and AAV8 capsid proteins. Baculovirus, a large DNA virus, serves as a helper virus for AAV in insect cells, producing AAV virion with the transfer DNA. Co-infected Sf9 cells were harvested 72 hours post-infection. Recombinant AAV8 (AAV8-TR2-OriP- mCardinal) was purified with iodixanol gradient ultracentrifugation for isolation (Figure 17A). With the baculovirus-mediated AAV preparation, we routinely isolated approximately 2 mL of 1013copies / ml of recombinant AAVs from 100 mL of Sf9 suspension culture. The purity of AAVs was monitored by SDS-PAGE gels with capsid protein bands as indicators, and highly pure fractions (i.e., fractions 1-4) were combined for use in experiments (Figure 17B, left). The frequencies of empty capsids that would impair transduction efficiency were also monitored by electron microscopy (Figure 17C). The result showed that more than 90% of purified AAV virions contained transfer DNAs.

[0183] With purified recombinant AAVs in our hand, we next transduced the AAV8 TR2-(?rzP-mCardinal to 293 / KSHV cells or the parental 293 cells and examined the amount of mCardinal expression with brightness. In AAV-transduced cells, the single- stranded AAVtransfer genomes become double- stranded circular DNA, like KSHV episome. Consistent with plasmid transfection (Figures 10C and 10D), mCardinal signals were significantly brighter in 293 / KSHV cells than parental 293 cells (Figure 17D and 17E, left). To rule out the possibility that the AAV infection efficiency is different between KSHV-infected and non-infected cells, we measured intracellular AAV DNA after infection. The results confirmed no differences in AAV infectivity (Figure 17E, right). Genomic copy measurement showed that KSHV infected cells maintained transduced vectors slightly better for four days (Figure 17F). However, majority of AAV vectors were eliminated at day 6 regardless of KSHV infection status. These results indicated that the major reason for selective gene expression is a KSHV infection specific promoter activation rather increased tethering of the transduced vector.(3) Selection of thymidine kinase gene.

[0184] Having vectors that preferentially express the exogenous genes in KSHV-infected cells, we next replaced mCardinal with a therapeutic gene. We selected the thymidine kinase (TK) / ganciclovir (GCV) for an indirect gene therapy. This is because KSHV is a herpesvirus, and GCV alone has been shown to control KSHV-associated tumor progression. Having a step for the conversion of the prodrug into a cytotoxic drug within the tumor cells makes the therapy adjustable. The converted cytotoxic compound is also reported to have a bystander effect of killing surrounding non-transduced tumor cells; this minimizes the necessity to transduce the AAV8- TR2-OriP-TK to 100% of tumor cells to be effective. We employed TKSR39, an engineered form of TK, which improved GCV conversion approximately ten times. The codon optimization for humans has also been reported to increase cell killing. Accordingly, we designed and synthesized the modified TK gene (Table 2) and purified recombinant AAVs with the baculovirus-based AAV production platform (Figure 2B, right).(4) AAV8-TR2-OriP-TK with Ganciclovir (GCV) induced cancer cell death in a KSHV infection-specific manner.

[0185] Next, we examined the degree to which the transduction of AAV8-TR2-OriP-TK specifically induces KSHV-infected cell death while sparing non-KSHV-infected cells. Preserving non-KSHV-infected cells should minimize side effects and increase the therapeutic window. We first used two KSHV-infected cell lines (293 cells and iSLK cells) and parental non-infected cells to consider the cell line bias. KSHV-infected (+) and non-infected (-) cells were transduced with AAV8-TR2-OriP-TK. Twenty-four hours post-transduction, we incubated with GCV (5 pM) with or without OTX015 (BRD4 inhibitor). Previous reports showed that theBRD4 inhibitor induces KSHV reactivation. Therefore, we expected OTX015 would increase TK expression from AAV therapeutic vectors. The results showed that transduction of AAV8- TR2-OriP-TK induced cell death only in the presence of GCV in both 293 and iSLK cells (Figures 18A and 18C). More importantly, the presence of latently infected KSHV strongly sensitized to the AAV8-TR2-OriP-TK (Figures 18B and 18D). In the case of 293 cells, the presence of KSHV is necessary to induce cell death with the AAV8-TR2-OriP-TK and GCV; the results indicate that the TR2-OriP activation is strictly regulated by KSHV infection (Figures 18A and 18B). Decreased cell viability in iSLK cells without KSHV infection may be due to leaky expression of K-Rta from exogenous K-Rta cassette, which can induce TK expression from the AAV vector. Figure 18E shows the morphology of the iSLK / KSHV and iSLK cells 2 days after AAV8-TR2-OriP-TK infection. The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA [i.e., vorinostat]), is known to trigger KSHV reactivation, and is an FDA-approved anti-cancer drug. We expected that the anti-cancer drugs that trigger KSHV reactivation would stimulate TR2-OriP transcription activity and, therefore, enhance the killing of cancer cells. OTX015 or SAHA were then incubated with GCV to examine the synergistic cell killing and proliferation. As we expected, SAHA and OTX015 enhanced TR2-OriP mediated gene expression and inhibited KSHV-infected cell growth.(5) AAV8-TR2-OriP-TK specifically inhibits the growth of KSHV- infected ECFCs.

[0186] While 293 and iSLK cells are important research tools for KSHV infection and report differences in sensitivity to the vector, those kidney cells are unlikely to be natural targets in KS patients. Endothelial colony-forming cells (ECFCs) may be the origin of KS. To examine if our vector is also effective in KSHV-infected ECFCs, we differentiated induced pluripotent stem cells (iPSCs) into ECFCs by following previous studies (Figure 19A). The differentiation of ECFCs was confirmed by the upregulation of ECFC markers, such as CD34, Prox-1, Flt-4, and LYVE-1, while the expression of the pluripotency markers, Oct3 / 4, Nanog, and Sox2, were significantly reduced with cell differentiation (Figure 19B). Consistent with previous studies, KSHV efficiently infected ECFCs but not iPSCs (Figure 19C). KSHV-infected ECFCs or parental ECFCs were transduced with AAV8-TR2-OriP-TK and treated with GCV (5 μtM) the following day. Live cells were counted over a three-day period. The results showed that AAV8- TR2-OriP-TK with GCV strongly induced KSHV-infected cell death, whereas uninfected ECFCs were not affected (Figures 19D and 19E). Fluorescent and bright-field imaging furtherconfirmed that the induction of cell death strictly depended on KSHV infection and GCV incubation (Figures 19D and 19E).(6) AAV8-TR2-OriP-TK prevents KSHV replication during reactivation.

[0187] One of the benefits of using GCV is that it inhibits KSHV DNA replication in reactivating cells, therefore preventing reactivated KSHV from infecting neighboring cells. To examine if AAV8-TR2-OriP-TK synergizes with endogenous KSHV TK and inhibits KSHV replication in the presence of GCV, we reactivated KSHV from iSLK cells with a combination of doxycycline with sodium butyrate and measured the effects of transduced AAV8-TR2-OriP- TK on KSHV replication (Figure 20A). This tactic is similar to delivering a "Trojan's horse" to the KSHV residence. The results showed that in the delivery of AAV8- TR2-OriP-TK in KSHV-infected cells, GCV abolished K8.1 late gene expression more than 100-fold, while PAN-RNA expression decreased approximately 10-fold (Figure 20B), which is consistent with the weaker RFP signal in A A V8-TR2-O / - / P-TK infected iSLK / KSHV cells (Figure 20A). The K8.1, a late gene expression, depends on the viral DNA replication, indicating that induction of TK from the AAV8-TR2-OriP-TK vector inhibited KSHV DNA replication. With additional TK, GCV also reduced KSHV virion production in culture media by approximately 10-fold (Figure 20C). Consequently, AAV8-TR2-OriP-TK reduced KSHV infections from 90% to 7% when the culture supernatant was transferred to freshly prepared iSLK cells (Figures 20D and 20E). These results indicate that AAV8-TR2-OriP-TK selectively inhibits KSHV-infected cell growth and prevents the spread of KSHV infection from reactivating cells.(7) Bystander effect of AAV8-TR2-OriP-TK in KSHV-infected cell population.

[0188] One of the bottlenecks of gene therapy is the efficacy of gene delivery. Even with the topical transduction of AAV8-TR2-OriP TK to KS skin or oral KS lesions with microneedles, we may not achieve 100% transduction efficacies. Thus, having the bystander cell-killing effect is critical. To monitor the bystander cell killing, we first prepared a recombinant BAC16 virus, which constitutively expresses the mCherry-H2B gene under the EFl alpha promoter. We replaced EGFP genes encoded in the BAC16 backbone with the mCherry-H2B coding sequence. The iSLK / KSHV BAC16 cells (EGFP-positive) were transduced AAV8-TR2-OriP-TK and cocultured with iSLK / mCherry-KSHV cells (without AAV-transduction) at 1:1 ratio, followed by GCV treatment for three days (Figure 21A). The results showed that GCV incubation inducedcell death not only in EGFP -positive cells (AAV-infected) but also in mCherry-positive cells (bystander cells), demonstrating the bystander effect of AAV-TK infection (Figure 21B).(8) AAV8-TR2-OriP-TK with GCV induces cell apoptosis.

[0189] To understand the mechanism of KSHV-infected cell killing, we next performed transcription profiling. By understanding the cell reactions to the vector, we can further enhance the cancer cell-killing effects with combination therapies. We extracted total RNA after 48 hours of GCV incubation and performed RNA-sequence. PCA analysis demonstrated that AAV8-TR2- OriP-TK. infection showed a very similar transcriptional profile in the absence of GCV in iSLK / KSHV and 293 / KSHV cells (Figure 21C). We used two pairs of cell lines to identify the signaling pathways commonly regulated by AAV8-TR2-OriP-TK under GCV. DAVID analysis for comprehensive functional annotation showed that DNA damage response and apoptotic process were enriched in iSLK cells transduced with AAV8-TR2-OriP-TK with GCV, while DNA damage response was the most enriched in 293 cells (Figure 21D). We also confirmed the presence of cleaved caspase 3 with GCV in AAV8-TR2-OriP-TK transduced cells (Figure 21E). These results indicate that AAV8-TR2-OriP-TK induced cell apoptosis with GCV via induction of DNA damage, and small molecule drugs that target DNA damage repair can have synergistic cancer cell killing effects. These results were consistent with previous reports that showed induction of cell apoptosis in other cell types with TK / GCV.(9) Anti-tumor activity in xenograft mice.

[0190] We finally examined the efficacy of cancer cell killing and toxicities in a xenograft mouse model. We first infected KSHV-infected iSLK cells with AAV8-TR2-OriP-TK in the culture dish and expanded the cells. Four days after AAV8-TR2-OriP-TK transduction, iSLK cells were harvested, and 5 xlO6cells were implanted at the subcutaneous of male NRG mice (right hind leg). No- AAV transduced cells were used as a comparison (left hind leg). GCV or PBS (50 mg / kg, twice a day) was administrated intraperitoneal (IP) for five days starting from two days after iSLK cells SQ injection (Figure 22A). As shown in Figure 22B, tumor mass was significantly diminished with GCV treatment. The tumor growth inhibition was AAV8-TR2- OriP- TK transduction and GCV administration specific because iSLK / KSHV cells without AAV8-TR2-OriP-TK planted in the same mice with GCV continued to grow. Similarly, without GCV treatment, AAV8-TR2-OriP-TK failed to inhibit tumor growth (Figures 22C, 22D, and 22E). As expected with a clinical drug, the GCV-treated mice did not show signs of discomfort or weight loss during the experiment periods. Immunohistochemistry with Ki67 antibodyAttorney Docket Number 11716-025WO1 showed that AAV8-TR2-OriP-TK with GCV strongly inhibited iSLK cell growth, and the overall number of KSHV-infected live cells (EGFP-positive) in the tumor mass is also lower (Figure 22F).(10) KSHV infection renders the resident cancer cell killing in xenograft mice

[0191] In vitro studies showed that KSHV infection is necessary for AAV8-TR2-OriP-TK to induce cell death in the presence of GCV. To further confirm the effects in the xenograft model, we first generated KSHV-infected Caki-1 cells, the origin of SLK cells. KSHV-infected and non- infected SLK cells (without K-Rta inducible cassette) were transduced with AAV8- TR2-OriP-TK, and AAV-transduced cells were implanted in mice (Figure 23 A). One group was treated with GCV, and the other group was treated with PBS. As expected from in vitro studies, KSHV infection made the cancer cells susceptible to AAV8-TR2-OriP-TK (Figure 23B-E). The results showed that non-KSHV infected SLK cells were able to form tumor masses despite the presence of AAV8-TR2-OriP-TK and GCV in the same mice (Figure 23 B, left panel).

[0192] Finally, we evaluated the toxicity of AAV8-TR2-OriP-TK with GCV after systemic injection. Since AAV8 preferentially targets the liver with systemic administration, we focused on assessing potential liver damage associated with GCV treatment. We first injected AAV8-TR2-OriP-TK systemically via IV and subsequently administrated GCV (50 mg / kg / twice a day) for five days. Twenty-four hours after the last GCV injections, organs and serum were harvested, and organ damages were examined with serum chemistry. The results showed no significant differences between the AAV8-TR2-OriP-TK with the GCV-treated group and the control group. b) Discussion

[0193] The incidence of KS in HIV+ patients has decreased since the introduction of antiretroviral therapy; however, KS remains the most common HIV-associated malignancy. Cancers with viral etiology, like KS, have more apparent therapeutic targets because the malignant cells usually express viral proteins, and those viral proteins are often responsible for cancer cell growth. Rapid LANA protein knock-down also led to the elimination of the KSHV genome from the cancer cells. However, finding small molecules to target LANA function effectively is still a work in progress in the KSHV research community.

[0194] Instead of targeting LANA directly, we took advantage of LANA function and well- developed KSHV transcription program to target KSHV-infected cells. The idea is based on a decade of KSHV gene regulation studies that showed that KSHV appears to establish atranscription program, which minimizes effects from cellular signaling events via well-designed enhancer and promoter DNA sequences that are primarily regulated by KSHV proteins. The relatively “closed” transcription regulatory mechanism designed for KSHV proteins allowed us to generate a gene expression vector whose expression is restricted in KSHV-infected cells. Based on previous KSHV promoter screening, we selected the Ori-RNA promoter, which is a direct target of K-Rta. We also showed that the Ori-RNA promoter is recruited by the LANA protein complex, presumably regulated by the LANA protein complex assembled at the TR region (enhancer region). Accordingly, by physically neighboring two genomic regions (TR and Ori-RNA promoter) by design, we expected to argue / tighten the LANA / TR-mediated transcription regulation, which we expected to make the transcription regulation more specific to KSHV infection.

[0195] Although increasing the number of TR copies in the vector is expected to enhance inducible promoter activity and improve plasmid tethering to host chromatins, we selected two copies in this study due to the limited space in the AAV vector and stability of plasmid DNA. We observed that plasmids with higher TR copy numbers frequently underwent recombination during amplification in E. coli, which may pose a problem when we increase the scale of production for clinical use. Identifying essential elements within the TR unit to shorten its sequence may offer a strategy to further enhance the vector efficacy.

[0196] Initially, we were concerned that the Ori-RNA promoter, although the strongest among KSHV lytic inducible promoters at the basal level, might still be insufficient to drive therapeutic gene expression to levels required for inducing cancer cell death. However, the weaker promoter worked positively by sparing non-KSHV-infected cells. Nonetheless, having several building blocks that include CMV gene enhancer element, other KSHV lytic gene promoters, and selection of therapeutic genes, we can flexibly design and generate multiple therapeutic vectors tailored to different applications. To evaluate our gene therapy approach, we first searched for cell lines that could infect both AAV8 and KSHV effectively and that were also used widely in the KSHV research community. The screening found that SLK cells and 293 cells were susceptible to KSHV and AAV8 infections. Later, we confirmed the efficacies of the approach in more clinically relevant ECFC. However, we also found that the KSHV naturally- infected primary effusion lymphoma cells (BC1, BC3, and BCBL-1) were refractory to AAV8 infection, indicating that additional capsid engineering or other AAV serotype's capsid needs to be used when we target PEL cells. Accordingly, our vector is primarily for skin and oral KS.Targeting cutaneous and oral KS with topical approaches provides a key advantage for gene therapy: direct and localized delivery. We should be able to administrate topically with microneedles or band-aids with hydro-gels to inject directly into tumors. With direct tumor injection, host immune response to the AAV capsid can enhance cancer cell killing. Alternatively, using mini-plasmid DNAs can reduce the risk of a host immune response, while increasing the shelf life of the drug.

[0197] We demonstrated two important advantages of applying the AAV8-TR2-OriP-TK to KSHV-associated malignancies: the specific killing of KSHV-infected cells and the inhibition of KSHV replication in reactivating cells. Importantly, GCV alone has already been proven to control KSHV-associated disease progression in clinics, presumably by inhibiting of viral spread from spontaneous reactivation, and subsequently suppressing inflammatory cytokine expression. We expect that the additional TK expression from AA V8-TR2-OriP-TK in KSHV-infected cancer cells should enhance the GCV effects locally and further widen the therapeutic windows to control KSHV-associated diseases.

[0198] Several anti-cancer drugs are known to reactivate KSHV in latently-infected cancer cells. The oncolytic strategy, which stimulates KSHV reactivation via an anti-cancer drug to kill cancer cells synergistically, has been examined in clinical trials. The hurdle of the oncolytic approach is to reactivate KSHV in all of the latently infected cancer cells. Transient expression of K-Rta with the stimulus does not always lead to a complete cycle of KSHV lytic replication. We showed enhanced cancer cell killing by AAV8-TR2-OriP-TK, along with bystander effects, when only 50% of the cell population in the dish was transduced with the vector. We expect that having the vector as an adjuvant should improve the outcome of the chemotherapy, especially for oncolytic therapy, which relies on small molecular drugs to stimulate K-Rta expression in the cancer cells.

[0199] In our mouse xenograft study, we transduced KSHV-infected iSLK cells with AAV8- TR2-OriP-TK in vitro and expanded these cells prior to subcutaneous implantation in NRG mice, resulting in near-uniform transduction of the cancer cells. The approach, however, does not reflect the challenges of delivering gene therapeutics to established solid tumors in a clinical setting. Solid tumors are characterized by barriers such as the tumor microenvironment and stromal components, which would significantly impede uniform AAV8 vector penetration and transgene expression, particularly deep within the tumor. By pre-transducing the cells, our invivo studies bypass these critical barriers, which limits the ability to evaluate the therapeutic feasibility in a more clinically relevant context.

[0200] In summary, based on a decade of foundational research on KSHV gene transcription, we designed, developed, and validated a KSHV disease- specific gene therapy vector. This vector, functioning as a KSHV-specific Trojan Horse, can expand the therapeutic window and alleviate the burden of chemotherapeutic side effects and the devastating impact of KSHV-associated diseases c) Materials and Methods(1) Chemicals, reagents, and antibodies

[0201] Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), phosphate-buffered saline (PBS), Trypsin-EDTA solution, 100 X penicillin-streptomycin-L-glutamine solution, Alexa 405-conjugated secondary antibody, Alexa 555 -conjugated secondary antibody, Alexa 647-conjugated secondary antibody, SlowFade Gold anti-fade reagent, Lipofectamine 2000 reagent, and high-capacity cDNA reverse transcription kit were purchased from Thermo Fisher (Waltham, MA, USA). Puromycin and G418 solution were obtained from InvivoGen (San Diego, CA, USA). Hygromycin B solution was purchased from Enzo Life Science (Farmingdale, NY, USA). Herpes simplex virus type 1 / 2 thymidine kinase monoclonal antibody was purchased from Invitrogen (San Diego, CA, USA). The Quick-RNA Miniprep kit was purchased from Zymo Research (Irvine, CA, USA), and the QIAamp DNA mini kit was purchased from QIAGEN (Germantown, MD, USA).(2) Cell culture

[0202] iSLK, Capi-1 (SLK cells), and 293 cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin-L-glutamine solution in 37°C, and 5% CO2. iSLK cells were obtained from Dr. Don Ganem (Novartis Institute for Biomedical Research) and cultured under 1 pg / ml puromycin. iSLK / KSHV, Capi-l / KSHV, and 293 / KSHV cells were also maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin-L-glutamine solution under 1 pg / ml puromycin and 500 pg / ml hygromycin. For reactivation, iSLK / KSHV cells were cultured for 4 or 5 days in the presence of 1 pM sodium butyrate and 1 pg / mL doxycycline in DMEM. Insect Sf9 cells were maintained in serum-free PSFM-J1 medium with 50 mL suspension cultures (Fuji Film Wako Chemical). iPSCs were generated from peripheral blood mononuclear cells by introducing the Yamanaka factors (OCT4, SOX2, KLF4, and c- MYC) using episomal vectors. After transduction, the cells were cultured in Stemfit (Ajinomoto,Japan) on iMatrix-511 -coated plates under hypoxic conditions (5% O2) at 37°C. Colonies with iPSC-like morphology were manually picked and expanded.(3) ECFC differentiation from iPSCs

[0203] After 2 days of culture in Stemfit media, iPSCs were stimulated with activin A (10 ng / mL) in the presence of FGF-2, VEGF 165, and BMP4 (10 ng / mL) for 24 hours. The following day, the media with cytokines was removed and replaced with Stemline II complete media (Sigma) containing FGF-2, VEGF165, and BMP4 (10 ng / ml) to promote endothelial cell emergence and expansion. Media was replaced with fresh Stemline II complete media on days 3, 5, 7, and 9. On day 12, the media was aspirated, and three parts of EGM-2 and one part of Stemline II complete media were added to the cultures. ECFC colonies appeared as tightly adherent cells.(4) Construction of pAAV-TR2-OriP-TK plasmid

[0204] A luciferase reporter plasmid, which encodes Ori-RNA promoter, was digested with restriction enzymes, Ncol and Xbal. The luciferase DNA fragment was replaced with a synthesized TKSR39 DNA fragment (IDTDNA), which also introduces Cpol restriction enzyme sites at the 5’ and 3' ends of the coding sequence. The cloning procedure also eliminates Xbal site. The DNA fragment encoding Ori-RNA promoter, TKSR39 coding sequence, and Poly(A) site were amplified with primers listed in Table 2, and cloned into pBlue Script TR2 plasmid at Kpnl-Spel site, which generates pTR2-OriP-TK. The AAV.CMV.PI.EGFP.WPRE.bGH plasmid was a gift from Dr. James M. Wilson (Addgene Plasmid #105530), and the plasmid was digested with restriction enzymes Nhel and BamHI, which removes CMV promoter and EGFP coding sequences. New multiple cloning sequences were prepared by annealing two single-stranded DNA (Table 2), and the resulting double-stranded DNA fragment was introduced to the AAV.CMV.PI.EGFP.WPRE.bGH Nhel-BamHl sites with Gene Assembly. The cloning generated unique sites for BamHI and Xbal in the pAAV.CMV.PI.EGFP.WPRE.bGH, resulting in a pAAV promoter / coding null vector. pTR2-OriP-TK was digested with Xbal and BamHI and cloned into a pAAV promoter / coding null vector, resulting in pA AV8-TR2-OriP-TK. Finally, SV40 poly (A) sites from the pGL3 vector were deleted with restriction enzyme digestion and swapped with WSPR.bGH, resulting in AAV.TR2.OriP.TK.WPRE.bGH. The TK coding sequence, which is cloned into Copl site, was exchanged with other Cpol DNA fragments encoding mCardinal and other reporter genes. To transfer the AAV transfer fragment into a Baculovirus transfer vector, pFAST-BACl (Invitrogen), was digested with BamHI and Hindllland blunted with T4 DNA polymerase. pAAV.TR2.OriP.TK.WPRE.bGH plasmid was digested with Pad, and the entire AAV transfer fragments including two inverted terminal repeats at both ends, were cloned into the pFAST-BAC vector blunted site. The cloning procedures are summarized in Figure IOC.(5) Preparation of recombinant baculoviruses with Bac to BAC system.

[0205] The pFAST-BAC transfer vector (pSR660), which expresses the bicistronic AAV-2 rep gene and polycistronic AAV-8 capsid genes, was a gift from Dr. Robert Kotin (Addgene Plasmid #65216). pFAST-BAC AAV.TR2.OriP.TK.WPRE.bGH or pSR660 were used to homologous recombination in BAC-to-BAC system in E.coli (invitrogen), and purified baculo virus DNAs from bacteria were transfected Sf9 cells. Recombinant baculoviruses were amplified twice and stored at 4°C for immediate use and at -80°C for stocks. Only passage 3 recombinant baculoviruses were used as working stocks to generate large-scale recombinant AAVs.(6) Preparation and purification of recombinant AAVs.

[0206] Sf9 cells (2.0 x 10s) cells were co-infected with recombinant baculoviruses encoding AAV transfer sequence and structure proteins and culture for 3 days in 50 mL suspension. Infected Sf9 cells were collected by centrifuge at 1,800g for 10 min at 4°C. Cell palettes were resuspended with resuspension buffer [50 mM Tris-HCl (pH 8.1), 150 mM NaCl, 2 mM MgCh]. Cells were lysis with four cycles of freeze and thaw, and lysed cells were incubated with Benzonase at 37°C for 1 hour. Cell lysates were centrifuged at 4,500 rpm for 15 min at 4 C. Supernatants were loaded on top of the iodixanol gradient. The step-wise gradient was centrifuged at 28,000 rpm for 16 hours at 4 °C with a SW28 rotor. The 40% iodixanol gradient was fractionated from bottom to top for every 500 uL, and the presence and purity of recombinant AAV were examined with 10% SDS-PAGE. A representative figure is shown in Figure 18B. Iodixanol fractions that contain AAV were pooled with Ami con Ultra Centrifuge Filter units (Millipore Sigma).(7) Quantification of KSHV copy number

[0207] The culture supernatant containing viral particles was treated with 1 mM MgCh and DNase I (12 iig / mL) for 15 minutes at room temperature. The reaction was stopped by the addition of EDTA to 5 mM, followed by heating at 70°C for 15 min. Viral genomic DNA waspurified using the QIAamp DNA Mini Kit according to the manufacturer’ s protocol and eluted in water. Elution was used for real-time qPCR to determine viral copy number.(8) Immunofluorescence Staining

[0208] Cells were seeded onto glass coverslips in a 6-well plate. After treatment, the cells were washed with PBS and fixed with 2% paraformaldehyde for 15 minutes at room temperature. The fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes and blocked with 5% bovine serum albumin (BSA) for 1 hour at 37°C. Primary antibodies (1:100 dilution) were diluted in a blocking buffer and incubated with the cells at 4 °C overnight. After washing three times with PBS, the cells were incubated with fluorophore- conjugated secondary antibodies (1:100 dilution) for 1 hour at room temperature in the dark. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature, followed by two additional washes with PBS. Coverslips were mounted onto glass slides using an anti-fade mounting medium. Fluorescence images were captured using a fluorescence microscope (BZ-X700), and image analysis was performed with BZ-X analyzer software.(9) RT-qPCR

[0209] iSLK cells 293 cells were infected with or without AAV for 2 days at MOI 104and 2.0 x 105cells were reseeded in 6 well plate. GCV, OTX105, and / or SAHA were added on the same day the cells were seeded and continued to culture at 37°C, 5% CO2. Total RNA was extracted using the Quick-RNA miniprep kit (Zymo Research, Irvine, CA, USA). A 500 ng of RNA was incubated with DNase I for 15 minutes and reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA USA). SYBR Green Universal master mix (Bio-Rad) was used for qPCR according to the manufacturer’s instructions. Each sample was normalized to 18S ribosomal RNA. All reactions were run in triplicate. Primer sequences used for qRT-PCR are provided in Table 2.(10) Flow cytometry

[0210] Cells were washed twice with PBS and resuspended in FACS buffer (PBS supplemented with 1% FBS). Flow cytometry was carried out by using a BD Acuri instrument (BD Biosciences), and data analysis was performed using FlowJo vlO.8.1 (Tree Star) by gating on live cells based on forward versus side scatter profiles.(11) Cryo-electron microscopy (Cryo-EM) imaging.

[0211] 4ul of each sample was applied to a glow-discharged (30mA, 30sec) holey carbon grid (300 mesh Quantifoil Rl.2 / 1.3 copper TEM grid) for plunge freezing in liquid nitrogen using Leica EM GP2 plunger at 18 °C. Cryo-EM images were acquired at 200 kV on a Thermo Scientific Glacios electron microscope equipped with a Gatan K3 direct electron detector. The micrographs were recorded at 56,818x (0.88 A / pixel) calibrated magnification using K3 with a dose of 40 e / AA2 and -1.6 um defocus using SerialEM.(12) RNA-sequencing

[0212] Indexed, stranded mRNA-seq libraries were prepared from total RNA (100 ng) using the KAPA Stranded mRNA-Seq kit (Roche) according to the manufacturer’s standard protocol. Libraries were pooled and multiplex sequenced on an Illumina NovaSeq 6000 System (150-bp, paired-end, >30 x 106reads per sample).

[0213] RNA-Seq data was analyzed using a Salmon-tximport-DESeq2 pipeline. Raw sequence reads (FASTQ format) were mapped to the reference human genome assembly (GRCh38 / hg38, GENCODE release 36) and quantified with Salmon. Gene-level counts were imported with tximport, and differential expression analysis, including Volcano plot, were performed with DESeq2.(13) Xenograft mouse model

[0214] All animal studies were conducted according to a UC Davis Institutional Animal Care and Use Committee (lACUC)-approved protocol. NRG (NOD.Cg-RagltmlMom I12rgtmlWjl / SzJ Strain #:007799) mouse breeding pairs were purchased from the Jackson Laboratory and the colony was maintained in-house.

[0215] 8-12-week-old NRG mice were injected subcutaneously with iSLK / KSHV cells, which were infected with or without AAV8-TR2-(OriP-TK for 4 days. Cells are resuspended in PBS and mixed with the same volume of Matrigel (Coming, #354230). One site (right or left hind leg) of each mouse was implanted 120pl containing 5 x 106cells. Mice were randomly assigned to PBS control or GCV groups. For the GCV group, each mouse was given 50 mg / kg GCV by intraperitoneal injection twice daily starting on day 2 and continued for 5 days. 50 mg / kg GCV were given twice per week staring from week 2. The tumor volume was measured and calculated as volume (mm3) = L xW2 / 2 (L is the largest diameter and W is the smallest diameter of the tumor) every three days. The experiment was terminated 38 days after the tumorcells implant. The mice were euthanized whenever the tumor size was over 20 mm, or the tumor volume was over 2000 mm3.(14) Statistical analysis

[0216] Statistical analyses were performed using GraphPad Prism 9.4.1 software. Results are shown as mean ± SD with dots representing individual measurements. Statistical significance was determined by Student’s t-test, ratio paired t-test, or one-way ANOVA with Tukey’s multiple comparison test, and correction for false discovery rate (FDR) as described in each figure legend. FDR corrected p < 0.05 was considered statistically significant.

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Claims

VII. CLAIMSWhat is claimed is:

1. A gene therapy vector comprising at least two copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) sequence, and a suicide gene operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell.

2. The gene therapy vector of claim 1, wherein the suicide gene is a tumor suppressor gene: TP53, activated caspase, cleaved poly(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, or p21.

3. The gene therapy vector of claim 1 or 2, wherein the vector is an adenovirus vector, adeno-associated virus (AAV) vector, lentivirus vector, or mini circle (DNA plasmid).

4. The gene therapy vector of claim 3, wherein the AAV vector is selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9.

5. The gene therapy vector of any one of claims 1-4, wherein the gene promoter comprises inducible gene promoter or tissue specific gene promoter.

6. The gene therapy vector of any one of claims 1-5, wherein the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, ORF57, LANA, vIL-6, KI, vGPCR, ORF74, or vFLIP.

7. The gene therapy vector of claim 1, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis.

8. The gene therapy vector of claim 7, wherein tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV trans activator.

9. The gene therapy vector of claim 8, wherein the KSHV transactivator activates the expression of therapeutic genes.

10. An anti-KSHV-infected malignancies therapy comprising the gene therapy vector of any one of claims 1-9 and an anti-herpes antiviral.

11. The anti-cancer therapy of claim 10, wherein the anti-herpes antiviral is selected from the group consisting of ganciclovir, acyclovir, valacyclovir, famciclovir, penciclovir, and valganciclovir.

12. A method of treating a KSHV-infected malignancies or disorders in a subject, comprising administering to the subject the gene therapy vector of any of claims 1-9 or the anti- KSHV-infected malignancies therapy of claim 10 or 11.

13. A method of treating a KSHV-infected malignancies or disorders in a subject, comprising administering to the subject a therapeutically effective amount of a gene therapy vector, wherein the gene therapy vector comprises at least two copies of the KSHV terminal repeat (TR) sequence, and a suicide gene operatively linked to a gene promoter; wherein the TR sequence enhances the expression of the suicide gene in a tumor cell.

14. The method of treating a KSHV-infected malignancies or disorders of claim 13, further comprising administering to the subject an anti-herpes antiviral.

15. The method of claim 14, wherein the anti-herpes antiviral is selected from the group consisting of ganciclovir, acyclovir, valacyclovir, famciclovir, penciclovir, and valganciclovir.

16. The method of any one of claims claim 12-15, wherein the KSHV-infected malignancies or disorders comprise Kaposi’s sarcoma, B cell lymphoma, Burkitt lymphoma, mantel cell lymphoma, human lymphoproliferative diseases, primary effusion lymphoma (PEL), AIDS- related multicentric Castleman’s disease (MCD), or KSHV-inflammatory cytokine syndrome (KICS).

17. The method of any one of claims 13-16, wherein the suicide gene is a tumor suppressor gene: TP53, activated caspase, cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP1), cytosine deaminase, purine nucleoside phophorylase (PNP), nitroreductase, guanine phosphorybosyl transferase, thymidine kinase (TK), carboxylesterases, cytochrome P450, or p21.

18. The method of any one of claims 13-17, wherein the gene therapy vector is an adenovirus vector, adeno-associated virus (AAV) vector, lentivirus vector, or mini circle (DNA plasmid).

19. The method of claim 18, wherein the AAV vector is selected from a group consisting of AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, and AAV9.

20. The method of any one of claims 13-19, wherein the gene promoter comprises an inducible gene promoter or tissue specific promoter.

21. The method of any one of claims 13-20, wherein the gene promoter comprises Ori-RNA, RTA, PAN RNA, vIL-6, LANA, KI, or ORF57, LANA, vIL-6, K12, vGPCR, ORF74, or vFLIP.

22. The method of any one of claims 13-21, wherein the gene promoter activates the expression of the suicide gene, wherein the suicide gene results in tumor cell apoptosis.

23. The method of any one of claims 13-22, wherein tumor cell apoptosis further reactivates KSHV, thereby activating the expression of KSHV trans activator.

24. The method of claim 23, wherein the KSHV transactivator activates the expression of therapeutic genes. TK, TP53, Caspases, Tumor necrosis factor (TNF)25. The method of any one of claims 13-24, further comprises administering to the subject a therapeutically effective amount of the gene therapy vector in combination with Suberoylanilide hydroxamic acid (SAHA), BET inhibitors, doxorubicin, etoposide, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), prednisone, or combination thereof.

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