Engineered lentivector targeting of dendritic cells for in vivo immunization

We report a method of inducing antigen production in dendritic cells by in vivo targeting with lentiviral vectors that specifically bind to the dendritic cell-surface protein DC-SIGN. To target dendritic cells, we enveloped the lentivector with a viral glycoprotein from Sindbis virus engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced dendritic cells and induced dendritic cell maturation. A high frequency (up to 12%) of ovalbumin (OVA)-specific CD8(+) T cells and a significant antibody response were observed 2 weeks after injection of a targeted lentiviral vector encoding an OVA transgene into naive mice. This approach also protected against the growth of OVA-expressing E.G7 tumors and induced regression of established tumors. Thus, lentiviral vectors targeting dendritic cells provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens.     

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.     

Cancer screening by systemic administration of a gene delivery vector encoding tumor-selective secretable biomarker expression

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a "theragnostic" agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.     

A new type of adenovirus vector that utilizes homologous recombination to achieve tumor-specific replication

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.     

Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18

We constructed pSin-SV40-HDV-SV40pA, an improved Sindbis DNA expression vector, and evaluated the potential of this vector system for brain tumor therapy. We investigated whether immunizing mice with xenogeneic DNA encoding human gp100 and mouse IL-18 would enhance the antitumor responses. To study the immune mechanisms involved in tumor regression, we examined tumor growth in B16-gp100-implanted brain tumor models using T-cell subset-depleted and IFN-gamma-neutralized mice. Hugp100/mIL-18 vaccination was also investigated for its antitumor effects against the wild-type murine B16 tumor, which expresses the murine gp100 molecule. Genetic immunization using plasmid pSin 9001 DNA codelivery of human gp100 and mouse IL-18 resulted in enhanced protective and therapeutic effects on the malignant brain tumors. The antitumor and protective effects were mediated by both CD4(+)/CD8(+) T cells and IFN-gamma. Vaccination with hugp100/mIL-18 conferred a significant survival merit to wild-type B16 tumor-harboring mice. Immunogene therapy with the improved Sindbis virus vector expressing xenogeneic gp100 and syngeneic IL-18 may be an excellent approach for developing a new treatment protocol. Thus, the Sindbis DNA system may represent a novel approach for the treatment of malignant brain tumors.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.     

Sindbis virus--an effective targeted cancer therapeutic

Viral therapies for cancer therapy have many potential positive attributes. These include the ability to specifically infect targeted cells, specifically express toxic or immune-enhancing genes, and the ability to specifically replicate within a tumor cell. Despite these biological advantages, efficacy to date has been limited. A recent report demonstrates that the Sindbis virus has remarkable properties in three challenging areas of gene therapy - specificity, efficacy and delivery, suggesting that Sindbis has the potential to become an important gene therapy vector for cancer therapy.     

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.     

DNA Immunization against Herpes Simplex Virus: Enhanced Efficacy Using a Sindbis Virus-Based Vector

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508–519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.     

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.     

Targeted Gene Delivery by Intravenous Injection of Retroviral Vectors

Specifically and effectively directing a therapeutic gene to its intended site of action is a critical issue for translation of basic genomics to clinical gene therapy. Delivering gene therapy vectors to specific cells or tissues through intravenous injection is the most desirable method for this purpose. In 2001, we reported successful targeted gene transduction in vitro utilizing both oncoretroviral and lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINDBIS). However, these pseudotypes mediated non-specific gene transduction to liver and spleen in vivo. To address this problem we generated the modified ZZ SINDBIS (termed m168) with significantly less non-specific infectivity. To investigate the ability of m168 pseudotyped lentiviral vector to mediate targeted gene transduction in vivo, we utilized a metastatic tumor model by using mouse melanoma cells engineered to express human P-glycoprotein. We administered the m168 pseudotyped vector conjugated with anti-P-glycoprotein antibody into the mice intravenously to target metastatic melanoma. The m168 pseudotyped vector selectively infected metastatic melanoma cells demonstrating successful targeted gene transduction in vivo. Targeting technology based upon m168 can be further modified for application not only to cancer but also potentially to genetic, neurologic, infectious and immune diseases, thereby expanding the future application of gene therapy.     

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.     

Tumor-targeted, systemic delivery of therapeutic viral vectors using hitchhiking on antigen-specific T cells

Antigen-specific T cells circulate freely and accumulate specifically at sites of antigen expression. To enhance the survival and targeting of systemically delivered viral vectors, we exploited the observation that retroviral particles adhere nonspecifically, or 'hitchhike,' to the surface of T cells. Adoptive transfer of antigen-specific T cells, loaded with viruses encoding interleukin (IL)-12 or Herpes Simplex Virus thymidine kinase (HSVtk), cured established metastatic disease where adoptive T-cell transfer alone was not effective. Productive hand off correlated with local heparanase expression either from malignant tumor cells and/or as a result of T-cell activation by antigen, providing high levels of selectivity for viral transfer to metastatic tumors in vivo. Protection, concentration and targeting of viruses by adsorption to cell carriers represent a new technique for systemic delivery of vectors, in fully immunocompetent hosts, for a variety of diseases in which delivery of genes may be therapeutically beneficial.     

Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors

Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.     

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.     

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.

To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.