HVS-based Vectors Research Articles

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.

A Herpesvirus saimiri-based vector expressing TRAIL induces cell death in human carcinoma cell lines and multicellular spheroid cultures

Herpesvirus saimiri (HVS) is capable of infecting a range of human carcinoma cell types with high efficiency and the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny upon cell division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. Herein we have produced a recombinant HVS-based vector containing full-length human TRAIL under the control of the α-survivin promoter, and subsequently challenged a variety of cancer cell lines with this vector. The TRAIL transgene was expressed in infected colorectal SW480 cells, causing considerable apoptosis induction. Apoptosis was also observed when several other cancer cell lines derived from different tissues were infected. Moreover, co-treatment with Jak inhibitor AG490 led to the disruption of spheroid cultures grown from the melanoma Mel888 line. These data suggest that an HVS gene therapy vector expressing TRAIL could be an effective treatment against cancer.

Production of Recombinant Herpesvirus Saimiri-Based Vectors: Figure 1.

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency. The viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny upon cell division. This allows HVS-based vectors to transduce stably a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome (BAC) cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to that of an artificial chromosome combined with an efficient delivery system. To insert and express a heterologous gene in an HVS-based vector, a recombinant virus must be constructed, as described in this protocol. An HVS-BAC is used to simplify and enhance the production of recombinant viruses. This requires a two-step process to insert the heterologous expression cassette first into the pHVS-Shuttle and then into the HVS-BAC.

Gardella Gel Analysis to Detect Herpesvirus Saimiri Episomal DNA

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency. The viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny on cell division. This allows HVS-based vectors to transduce stably a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome (BAC) cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to that of an artificial chromosome combined with an efficient delivery system. In this protocol, Gardella gel analysis is performed to determine whether the HVS genome is maintained in a nonintegrated episomal form. This method allows the identification of chromosomal/integrated DNA and episomal and linear forms of viral DNA, which run at the top, middle, and bottom of the gel, respectively. To obtain the required sensitivity to detect HVS episomes within host tissues, a modified polymerase chain reaction (PCR)-based Gardella gel can also be utilized.

Assessment of Infectivity Using a Herpesvirus Saimiri (HVS) Recombinant that Expresses HVS–GFP: Figure 1.

Herpesvirus saimiri (HVS) is capable of infecting a wide range of human cell types with high efficiency. The viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny on cell division. This allows HVS-based vectors to transduce stably a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome (BAC) cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to that of an artificial chromosome combined with an efficient delivery system. This protocol describes the use of an HVS recombinant virus expressing green fluorescent protein (GFP) (HVS-GFP) to assess the infectivity of a specific cell line.

Herpesvirus Saimiri-Based Gene Delivery Vectors

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.

Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Here we assess the dissemination of HVS-based vectors in vivo following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases.

Efficient infection and persistence of a herpesvirus saimiri-based gene delivery vector into human tumor xenografts and multicellular spheroid cultures

Herpesvirus saimiri (HVS) has the ability to infect a variety of human cell lines and establish a persistent infection by virtue of episomal maintenance. Moreover, the viral episome provides sustained expression of a heterologous transgene. HVS-based vectors can also persist for a long term in tumor xenografts generated from HVS-infected human carcinoma cell lines. The viral episome remains latent within the xenograft allowing long-term transgene expression. These properties, in addition to its ability to incorporate large amounts of heterologous DNA, make HVS an attractive potential gene delivery vector. Here we report on the further evaluation of such HVS-based vectors. We demonstrate for the first time that HVS can efficiently infect solid tumor xenografts derived from a variety of human carcinoma cells via direct intratumoral injections. Furthermore, HVS can efficiently infect spheroid cultures, a three-dimensional cell culture system that closely resembles a tumor. Upon infection of both the tumor xenografts and spheroid cultures, HVS-based vectors can establish a persistent episomal infection within the tumor xenograft allowing expression of a heterologous transgene. These results suggest that HVS-based vectors may be suitable for cancer gene therapy applications.

The herpesvirus saimiri ORF 73 regulatory region provides long-term transgene expression in human carcinoma cell lines.

Herpesvirus saimiri (HVS) is capable of establishing a persistent infection in a variety of human carcinoma cell lines, by virtue of episomal maintenance. Moreover, the viral episome provides expression of a transgene in both in vitro and in vivo environments. At present, HVS vectors utilize heterologous promoters such as the IE hCMV promoter. However, this promoter maybe unsuitable for long-term expression in vivo, as promoter silencing has been observed in this and other herpesvirus-based vector systems. Ideal regulatory regions would be functional when the herpesvirus genome is maintained as a latent episome. We have previously shown that gene expression in an HVS-persistently-infected human carcinoma cell line is limited to an adjacent set of genes encoding ORFs 71-73. These genes are transcribed as a polycistronic mRNA species from a common regulatory region upstream of the ORF 73 gene. In this report, we assess the potential of the ORF 73 regulatory region to provide heterologous gene expression in a wide variety of human cancer cell lines. We demonstrate, utilizing transient transfection assays, that the ORF 73 regulatory region can provide transgene expression in a variety of human carcinoma cell lines, although levels of transgene expression are not as high as achieved under the control of heterologous promoters such as the IE hCMV promoter. Furthermore, incorporation of the minimal ORF 73 regulatory region in a recombinant HVS-based vector provides sustained expression of the green fluorescent protein in both in vitro and in vivo environments. These results suggest that the ORF 73 regulatory region may be suitable for use in HVS-based cancer gene therapy applications.

In vivo episomal maintenance of a herpesvirus saimiri-based gene delivery vector.

Herpesvirus saimiri (HVS) has several properties that make it amenable to development as a gene delivery vector. HVS offers the potential to incorporate large amounts of heterologous DNA and infect a broad range of human cell lines. Upon infection the viral genome can persist by virtue of episomal maintenance and stably maintains heterologous gene expression. Here we report an evaluation of the in vivo properties of HVS, with a view to its development as a gene delivery system. We demonstrate for the first time, the long-term persistence of the HVS genome in tumour xenografts generated from HVS-infected human carcinoma cell lines. The HVS-based vector remained latent in the xenograft without spreading to other organs. Moreover, the long-term in vivo maintenance of the HVS genome, as a nonintegrated circular episome, provided efficient sustained expression of a heterologous transgene. These in vivo results suggest that HVS-based vectors have potential for gene therapy applications.

Stable marker gene transfer into human bone marrow stromal cells and their progenitors using novel herpesvirus saimiri-based vectors.

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.