Mark J. Federspiel

Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

ABSTRACT We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

Phase I Trial of Intraperitoneal Administration of an Oncolytic Measles Virus Strain Engineered to Express Carcinoembryonic Antigen for Recurrent Ovarian Cancer

Edmonston vaccine strains of measles virus (MV) have shown significant antitumor activity in preclinical models of ovarian cancer. We engineered MV to express the marker peptide carcinoembryonic antigen (MV-CEA virus) to also permit real-time monitoring of viral gene expression in tumors in the clinical setting. Patients with Taxol and platinum-refractory recurrent ovarian cancer and normal CEA levels were eligible for this phase I trial. Twenty-one patients were treated with MV-CEA i.p. every 4 weeks for up to 6 cycles at seven different dose levels (10(3)-10(9) TCID(50)). We observed no dose-limiting toxicity, treatment-induced immunosuppression, development of anti-CEA antibodies, increase in anti-MV antibody titers, or virus shedding in urine or saliva. Dose-dependent CEA elevation in peritoneal fluid and serum was observed. Immunohistochemical analysis of patient tumor specimens revealed overexpression of measles receptor CD46 in 13 of 15 patients. Best objective response was dose-dependent disease stabilization in 14 of 21 patients with a median duration of 92.5 days (range, 54-277 days). Five patients had significant decreases in CA-125 levels. Median survival of patients on study was 12.15 months (range, 1.3-38.4 months), comparing favorably to an expected median survival of 6 months in this patient population. Our findings indicate that i.p. administration of MV-CEA is well tolerated and results in dose-dependent biological activity in a cohort of heavily pretreated recurrent ovarian cancer patients.

Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer

Establishment of the mammalian germ line is a prerequisite for fertility of the adult animal but we know surprisingly little about the molecular mechanisms regulating germ-line development in mammals. Signaling from the c-Kit receptor tyrosine kinase is essential for primordial germ cell (PGC) growth both in vivo and in vitro. Many downstream effectors of the c-Kit signaling pathway have been identified in other cell types but how these molecules control PGC survival and proliferation are unknown. Determination of the c-Kit effectors acting in PGCs has been hampered by the lack of effective methods to easily manipulate gene expression in these cells. We overcame this problem by testing the efficacy of retroviral-mediated gene transfer for manipulating gene expression in mammalian germ cells. We found that PGCs can be successfully infected with a variety of types of retroviruses. We used this method to demonstrate an important role for the AKT kinase in regulating PGC growth. Such technology for manipulating gene expression in PGCs will allow many of the molecular mechanisms regulating germ cell growth, behavior, and differentiation to be comprehensively analyzed.

RETROVIRAL GENE DELIVERY

Publisher Summary This chapter describes the properties of retroviruses and the affect of these properties on the properties of retrovirus-based vectors. In addition, the advantages and disadvantages of retroviral vectors to those of several other gene transfer techniques are compared. The chapter describes the retroviral vector system based on the avian leukosis virus (ALV) retrovirus family. The ALV-based retroviral vector system can be used to deliver and express genes in avian and mammalian cells in vitro and in vivo. The system can be used to exploit the advantages of both the avian and murine animal models. Replication-competent ALV-based vectors can be grown to high titer on avian cells but are replication-defective after infecting susceptible mammalian cells. The system offers advantages for experiments that require either widespread or tissue-specific gene delivery. If the target cells continue to divide, multiple experimental genes can be delivered sequentially by multiple retroviral infections. Other gene-delivery methods could be used in combination with the ALV-based retroviral vector system to deliver a second gene in cells that have stopped dividing.

Preclinical Pharmacology and Toxicology of Intravenous MV-NIS, an Oncolytic Measles Virus Administered With or Without Cyclophosphamide

MV‐NIS is an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS). Here, we report the results of preclinical pharmacology and toxicology studies conducted in support of our clinical protocol “Phase I Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or Refractory Multiple Myeloma.” Dose–response studies in the KAS‐6/1 myeloma xenograft model demonstrated a minimum effective dose of 4 × 106 TCID50 (tissue culture infectious dose 50)/kg. Toxicity studies in measles‐naive squirrel monkeys and measles‐susceptible transgenic mice were negative at intravenous doses up to 108 and 4 × 108 TCID50/kg, respectively. Abundant viral mRNA, maximal on day 8, was detected in cheek swabs of squirrel monkeys, more so after pretreatment with cyclophosphamide. On the basis of these data, the safe starting dose of MV‐NIS for our clinical protocol was set at 1−2 × 104 TCID50/kg (106 TCID50 per patient).

Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma

The oncolytic measles virus Edmonston strain (MV‐Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV‐Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus‐infected cells. We evaluated recombinant MV‐Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV‐Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV‐Edm in vitro and in vivo was assessed by CEA concentration, 125I‐uptake, and 123I‐imaging studies. Toxicology studies were performed in IfnarKO×CD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV‐Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV‐Edm resulted in high‐level expression of transgenes in vitro and in vivo. MV‐Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV‐Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV‐Edm may be a potent and novel cancer gene therapy system for HCC. MV‐Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner. (HEPATOLOGY 2006;44:1465–1477.)

Dual Therapy of Ovarian Cancer Using Measles Viruses Expressing Carcinoembryonic Antigen and Sodium Iodide Symporter

Purpose: MV-CEA is an oncolytic measles virus currently being tested in patients with ovarian cancer and whose propagation can be monitored by measuring blood carcinoembryonic antigen (CEA) levels. MV-NIS is an oncolytic measles virus coding for the thyroidal sodium iodide symporter (NIS) whose propagation can be mapped by serial radioiodine imaging. Expression of both CEA and NIS genes from a single virus would combine sensitive, quantitative expression monitoring (CEA) with radioisotopic expression mapping (NIS). Because of the unfavorable replication kinetics of measles viruses expressing both CEA and NIS, we explored the feasibility of combining MV-CEA with MV-NIS for comprehensive virotherapy monitoring in ovarian cancer. Experimental Design and Results: Mice implanted with i.p. SKOV3ip.1 ovarian cancer xenografts received MV-CEA alone, MV-NIS alone, or a combination of MV-CEA plus MV-NIS. Viral gene expression was monitored by measuring blood CEA levels, and the location of virus-infected cells was monitored by gamma camera imaging. Surprisingly, mice receiving the combination of MV-CEA plus MV-NIS showed greatly superior responses to therapy, but this was associated with 10-fold lower plasma levels of CEA compared with mice treated with MV-CEA alone. In vitro studies showed superior replication kinetics of MV-NIS relative to MV-CEA. The gamma camera scans were considerably less sensitive than the plasma CEA marker for monitoring virus infection. Conclusions: Dual therapy with MV-CEA and MV-NIS is superior to treatment with either virus alone, and it allows noninvasive monitoring of virotherapy via soluble marker peptide and gamma camera imaging. This has important implications for the clinical development of oncolytic measles viruses.

Expression of IFN-β Enhances Both Efficacy and Safety of Oncolytic Vesicular Stomatitis Virus for Therapy of Mesothelioma

Our preclinical and clinical trials using a replication-defective adenoviral vector expressing IFN-beta have shown promising results for the treatment of malignant mesothelioma. Based on the hypotheses that a replication-competent vesicular stomatitis virus (VSV) oncolytic vector would transduce more tumor cells in vivo, that coexpression of the immunostimulatory IFN-beta gene would enhance the immune-based effector mechanisms associated both with regression of mesotheliomas and with VSV-mediated virotherapy, and that virus-derived IFN-beta would add further safety to the VSV platform, we tested the use of IFN-beta as a therapeutic transgene expressed from VSV as a novel treatment for mesothelioma. VSV-IFN-beta showed significant therapy against AB12 murine mesotheliomas in the context of both local and locoregional viral delivery. Biologically active IFN-beta expressed from VSV added significantly to therapy compared with VSV alone, dependent in part on host CD8+ T-cell responses. Immune monitoring suggested that these antitumor T-cell responses may be due to a generalized T-cell activation rather than the priming of tumor antigen-specific T-cell responses. Finally, IFN-beta also added considerable extra safety to the virus by providing protection from off-target viral replication in nontumor tissues and protected severe combined immunodeficient mice from developing lethal neurotoxicity. The enhanced therapeutic index provided by the addition of IFN-beta to VSV therefore provides a powerful justification for the development of this virus for future clinical trials.

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 ((125)I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of (123)I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 x 10(6) TCID(50)) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of (131)I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by (131)I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.

Optimizing patient derived mesenchymal stem cells as virus carriers for a Phase I clinical trial in ovarian cancer

BackgroundMesenchymal stem cells (MSC) can serve as carriers to deliver oncolytic measles virus (MV) to ovarian tumors. In preparation for a clinical trial to use MSC as MV carriers, we obtained cells from ovarian cancer patients and evaluated feasibility and safety of this approach.MethodsMSC from adipose tissues of healthy donors (hMSC) and nine ovarian cancer patients (ovMSC) were characterized for susceptibility to virus infection and tumor homing abilities.ResultsAdipose tissue (range 0.16-3.96 grams) from newly diagnosed and recurrent ovarian cancer patients yielded about 7.41×106 cells at passage 1 (range 4–9 days). Phenotype and doubling times of MSC were similar between ovarian patients and healthy controls. The time to harvest of 3.0×108 cells (clinical dose) could be achieved by day 14 (range, 9–17 days). Two of nine samples tested had an abnormal karyotype represented by trisomy 20. Despite receiving up to 1.6×109 MSC/kg, no tumors were seen in SCID beige mice and MSC did not promote the growth of SKOV3 human ovarian cancer cells in mice. The ovMSC migrated towards primary ovarian cancer samples in chemotaxis assays and to ovarian tumors in athymic mice. Using non-invasive SPECT-CT imaging, we saw rapid co-localization, within 5–8 minutes of intraperitoneal administration of MV infected MSC to the ovarian tumors. Importantly, MSC can be pre-infected with MV, stored in liquid nitrogen and thawed on the day of infusion into mice without loss of activity. MV infected MSC, but not virus alone, significantly prolonged the survival of measles immune ovarian cancer bearing animals.ConclusionsThese studies confirmed the feasibility of using patient derived MSC as carriers for oncolytic MV therapy. We propose an approach where MSC from ovarian cancer patients will be expanded, frozen and validated to ensure compliance with the release criteria. On the treatment day, the cells will be thawed, washed, mixed with virus, briefly centrifuged and incubated for 2 hours with virus prior to infusion of the virus/MSC cocktail into patients.