Justin C. Roth

Long-Term-Infected Telomerase-Immortalized Endothelial Cells: a Model for Kaposi's Sarcoma-Associated Herpesvirus Latency In Vitro and In Vivo

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is associated with Kaposis sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castlemans disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.

Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo

Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone.

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-Methylguanine-DNA Methyltransferase Mutants: Implications for Drug Resistance Gene Therapy

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.

Cell vehicle targeting strategies

Use of cells as therapeutic carriers has increased in the past few years and has developed as a distinct concept and delivery method. Cell-based vehicles are particularly attractive for delivery of biotherapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction. Initial studies using cell-based approaches served to identify some of the key factors for the success of this type of therapeutic delivery. These factors include the efficiency of cell loading with a therapeutic payload, the means of cell loading and the nature of therapeutics that cells can carry. However, one important aspect of cell-based delivery yet to be fully investigated is the process of actual delivery of the cell payload in vivo. In this regard, the potential ability of cell carriers to provide site-specific or targeted delivery of therapeutics deserves special attention. The present review focuses on a variety of targeting approaches that may be utilized to improve cell-based therapeutic delivery strategies. The different aspects of targeting that can be applied to cell vehicles will be discussed, including physical methods for directing cell distribution, intrinsic cell-mediated homing mechanisms and the feasibility of engineering cells with novel targeting mechanisms. Development of cell targeting strategies will further advance cell vehicle applications, broaden the applicability of this delivery approach and potentiate therapeutic outcomes.

Targeting of mesenchymal stem cells to ovarian tumors via an artificial receptor

BackgroundMesenchymal Progenitor/Stem Cells (MSC) respond to homing cues providing an important mechanism to deliver therapeutics to sites of injury and tumors. This property has been confirmed by many investigators, however, the efficiency of tumor homing needs to be improved for effective therapeutic delivery. We investigated the feasibility of enhancing MSC tumor targeting by expressing an artificial tumor-binding receptor on the MSC surface.MethodsHuman MSC expressing an artificial receptor that binds to erbB2, a tumor cell marker, were obtained by transduction with genetically modified adenoviral vectors encoding an artificial receptor (MSC-AR). MSC-AR properties were tested in vitro in cell binding assays and in vivo using two model systems: transient transgenic mice that express human erbB2 in the lungs and ovarian xenograft tumor model. The levels of luciferase-labeled MSCs in erbB2-expressing targeted sites were evaluated by measuring luciferase activity using luciferase assay and imaging.ResultsThe expression of AR enhanced binding of MSC-AR to erbB2-expressing cells in vitro, compared to unmodified MSCs. Furthermore, we have tested the properties of erbB2-targeted MSCs in vivo and demonstrated an increased retention of MSC-AR in lungs expressing erbB2. We have also confirmed increased numbers of erbB2-targeted MSCs in ovarian tumors, compared to unmodified MSC. The kinetic of tumor targeting by ip injected MSC was also investigated.ConclusionThese data demonstrate that targeting abilities of MSCs can be enhanced via introduction of artificial receptors. The application of this strategy for tumor cell-based delivery could increase a number of cell carriers in tumors and enhance efficacy of cell-based therapy.

Enhancement of antitumor immunity in lung cancer by targeting myeloid-derived suppressor cell pathways.

Chemoresistance due to heterogeneity of the tumor microenvironment (TME) hampers the long-term efficacy of first-line therapies for lung cancer. Current combination therapies for lung cancer provide only modest improvement in survival, implicating necessity for novel approaches that suppress malignant growth and stimulate long-term antitumor immunity. Oxidative stress in the TME promotes immunosuppression by tumor-infiltrating myeloid-derived suppressor cells (MDSC), which inhibit host protective antitumor immunity. Using a murine model of lung cancer, we demonstrate that a combination treatment with gemcitabine and a superoxide dismutase mimetic targets immunosuppressive MDSC in the TME and enhances the quantity and quality of both effector and memory CD8(+) T-cell responses. At the effector cell function level, the unique combination therapy targeting MDSC and redox signaling greatly enhanced cytolytic CD8(+) T-cell response and further decreased regulatory T cell infiltration. For long-term antitumor effects, this therapy altered the metabolism of memory cells with self-renewing phenotype and provided a preferential advantage for survival of memory subsets with long-term efficacy and persistence. Adoptive transfer of memory cells from this combination therapy prolonged survival of tumor-bearing recipients. Furthermore, the adoptively transferred memory cells responded to tumor rechallenge exerting long-term persistence. This approach offers a new paradigm to inhibit immunosuppression by direct targeting of MDSC function, to generate effector and persistent memory cells for tumor eradication, and to prevent lung cancer relapse.

Targeting EGFR with metabolically biotinylated fiber-mosaic adenovirus

Adenovirus (Ad)-based vectors are useful gene delivery vehicles for a variety of applications. Despite their attractive properties, many in vivo applications require modulation of the viral tropism. Targeting approaches applied to adenoviral vectors included genetic modification of the viral capsid, controlled expression of the transgene and combinatorial approaches that combine two or more targeting elements in single vectors. Most of these studies confirmed successful retargeting in cell cultures, however, in vivo gains of targeted adenoviral vectors have not been widely demonstrated. We have developed a combinatorial retargeting approach utilizing metabolically biotinylated Ad, where the biotin acceptor peptide was incorporated in one of the fibers in a dual fiber viral particle resulting in metabolically biotinylated fiber-mosaic Ad (mBfMAd). We have utilized this vector in complex with epidermal growth factor (EGF)-Streptavidin to retarget fiber-mosaic virus to EGF receptor (EGFR) expressing cells in vitro and confirmed an increased infectivity of the retargeting complex. Most importantly, the utility of this strategy was demonstrated in vivo in two distinct animal models. In both models tested, retargeted mBfMAd demonstrated an increased ratio of gene expression in target tissues compared to the liver expression profile. Thus, metabolically biotinylated fiber-mosaic virus in combination with appropriate adapters can be successfully exploited for adenoviral retargeting strategies.

Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.

Oncolytic viral therapy: targeting cancer stem cells

Cancer stem cells (CSCs) are defined as rare populations of tumor-initiating cancer cells that are capable of both self-renewal and differentiation. Extensive research is currently underway to develop therapeutics that target CSCs for cancer therapy, due to their critical role in tumorigenesis, as well as their resistance to chemotherapy and radiotherapy. To this end, oncolytic viruses targeting unique CSC markers, signaling pathways, or the pro-tumor CSC niche offer promising potential as CSCs-destroying agents/therapeutics. We provide a summary of existing knowledge on the biology of CSCs, including their markers and their niche thought to comprise the tumor microenvironment, and then we provide a critical analysis of the potential for targeting CSCs with oncolytic viruses, including herpes simplex virus-1, adenovirus, measles virus, reovirus, and vaccinia virus. Specifically, we review current literature regarding first-generation oncolytic viruses with their innate ability to replicate in CSCs, as well as second-generation viruses engineered to enhance the oncolytic effect and CSC-targeting through transgene expression.

Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a conditionally-replicating oncolytic HSV-1.

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x107 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.