Suppression of bone metastatic castration-resistant prostate cancer cell growth by a suicide gene delivered by JC polyomavirus-like particles.

Prostate cancer is one of the most common cancers in men. The heterogeneity and mutations exhibited by prostate cancer cells often results in the progression to incurable metastatic castration-resistant prostate cancer (mCRPC). Our previous investigations demonstrated that the virus-like particles (VLPs) of JC polyomavirus (JCPyV) can deliver exogenous genes to prostate cancer cells for expression. JCPyV VLPs packaging pPSAtk (PSAtk-VLPs) possess the ability to transcriptionally target and selectively induce cytotoxicity in prostate cancer cells in vitro and in vivo, as pPSAtk can only express the thymidine kinase gene, a suicide gene, in androgen receptor-positive cells. To further investigate whether PSAtk-VLPs inhibit the growth of metastasized prostate cancer cells, we established an animal model of bone-metastatic prostate cancer to compare PSAtk-VLPs with leuprorelin acetate and enzalutamide, hormonal agents commonly used in clinical settings, and investigated the effectiveness of PSAtk-VLPs. In the present study, we observed that PSAtk-VLPs effectively inhibited the growth of prostate cancer cells that had metastasized to the bone in the metastatic animal model. In addition, PSAtk-VLPs showed a higher effectiveness than hormone therapy in this animal model study. These results suggest that PSAtk-VLPs may serve as a treatment option for mCRPC therapy in the future.

Inhibition of human lung adenocarcinoma growth and metastasis by JC polyomavirus-like particles packaged with an SP-B promoter-driven CD59-specific shRNA.

Lung cancer ranks first in both incidence and mortality and is a major health concern worldwide. Upon recognition of specific antigens on tumor cells, complement-dependent cytotoxicity (CDC) is activated, arresting cell growth or inducing apoptosis. However, by overexpressing CD59, a membrane complement regulatory protein (mCRP), lung cancer cells develop resistance to CDC. We previously showed that virus-like particles (VLPs) of human JC polyomavirus (JCPyV) could be used as a gene therapy vector to carry a suicide gene expression plasmid with a lung-specific promoter (SP-B (surfactant protein B)) for lung adenocarcinomas. Herein, we designed a CD59-specific short hairpin RNA (shRNA) expression plasmid driven by SP-B (pSPB-shCD59) to effectively and specifically inhibit CD59 overexpression in lung cancer cells. Treatment of lung cancer cells in vitro with JCPyV VLPs containing pSPB-shCD59 (pSPB-shCD59/VLPs) induces CDC and death of cancer cells. Mice that were subcutaneously injected with human lung cancer cells showed an 87% inhibition in tumor growth after tail vein injection of pSPB-shCD59/VLPs. Moreover, in a mouse model of lung cancer metastasis, a reduction in the lung weight by 39%, compared with the control group, was observed in mice treated with pSPB-shCD59/VLPs after tail vein injection of human lung cancer cells. Furthermore, tissue sectioning showed that the number and size of tumors produced was significantly reduced in the lungs of mice in the treatment group than those of the untreated group, indicating inhibition of metastasis by pSPB-shCD59/VLPs. Together, these results demonstrate the potential of pSPB-shCD59/VLPs as a therapeutic agent for CD59 overexpressed lung cancer.

Inhibition of human diffuse large B-cell lymphoma growth by JC polyomavirus-like particles delivering a suicide gene.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of aggressive B-cell non-Hodgkin lymphoma. About one-third of patients are either refractory to the treatment or experience relapse afterwards, pointing to the necessity of developing other effective therapies for DLBCL. Human B-lymphocytes are susceptible to JC polyomavirus (JCPyV) infection, and JCPyV virus-like particles (VLPs) can effectively deliver exogenous genes to susceptible cells for expression, suggesting the feasibility of using JCPyV VLPs as gene therapy vectors for DLBCL. METHODS: The JCPyV VLPs packaged with a GFP reporter gene were used to infect human DLBCL cells for gene delivery assay. Furthermore, we packaged JCPyV VLPs with a suicide gene encoding thymidine kinase (TK) to inhibit the growth of DLBCL in vitro and in vivo. RESULTS: Here, we show that JCPyV VLPs effectively entered human germinal center B-cell-like (GCB-like) DLBCL and activated B-cell-like (ABC-like) DLBCL and expressed the packaged reporter gene in vitro. As measured by the MTT assay, treatment with tk-VLPs in combination with gancyclovir (GCV) reduced the viability of DLBCL cells by 60%. In the xenograft mouse model, injection of tk-VLPs through the tail vein in combination with GCV administration resulted in a potent 80% inhibition of DLBCL tumor nodule growth. CONCLUSIONS: Our results demonstrate the effectiveness of JCPyV VLPs as gene therapy vectors for human DLBCL and provide a potential new strategy for the treatment of DLBCL.

Inhibition of human bladder cancer growth by a suicide gene delivered by JC polyomavirus virus-like particles in a mouse model.

PURPOSE: Bladder cancer is one of the most common cancers of the urinary tract. The poor 5-year survival rate of invasive bladder cancer represents a challenge for bladder cancer treatment. Previous studies demonstrated that human urothelial carcinoma is susceptible to infection by JC polyomavirus. We used JC polyomavirus virus-like particles to deliver genes into human urothelial carcinoma cells for possible therapeutic investigation. MATERIALS AND METHODS: Reporter plasmids (pEGFP-N3) for expressing green fluorescent protein, LacZ expression plasmids bearing cytomegalovirus or Muc1 promoter and a functional plasmid (pUMVC1-tk) for expressing thymidine kinase were packaged into JC polyomavirus virus-like particles. Plasmid DNAs were transduced via the JC polyomavirus virus-like particles into human urothelial carcinoma cells in vitro and into xenografted human bladder tumor nodules in vivo. RESULTS: pEGFP-N3 DNA was delivered and green fluorescent protein was expressed in human urothelial carcinoma cells in vitro and in the tumor nodules of mice in vivo. The thymidine kinase transgene also functioned in vitro and in vivo after JC polyomavirus virus-like particle transduction. The thymidine kinase gene transduced urothelial carcinoma nodules were drastically reduced in the presence of acyclovir. In addition, we noted selective Muc1-LacZ expression in human urothelial carcinoma cells transduced by JC polyomavirus virus-like particles. CONCLUSIONS: These findings provide a possible future approach to human urothelial carcinoma gene therapy using JC polyomavirus virus-like particles.

Recombined sequences between the non-coding control regions of JC and BK viruses found in the urine of a renal transplantation patient.

Kidney cells are the common host for JC virus (JCV) and BK virus (BKV). Reactivation of JCV and/or BKV in patients after organ transplantation, such as renal transplantation, may cause hemorrhagic cystitis and polyomavirus-associated nephropathy. Furthermore, JCV and BKV may be shed in the urine after reactivation in the kidney. Rearranged as well as archetypal non-coding control regions (NCCRs) of JCV and BKV have been frequently identified in human samples. In this study, three JC/BK recombined NCCR sequences were identified in the urine of a patient who had undergone renal transplantation. They were designated as JC-BK hybrids 1, 2, and 3. The three JC/BK recombinant NCCRs contain up-stream JCV as well as down-stream BKV sequences. Deletions of both JCV and BKV sequences were found in these recombined NCCRs. Recombination of DNA sequences between JCV and BKV may occur during co-infection due to the relatively high homology of the two viral genomes.

Peptide-guided JC polyomavirus-like particles specifically target bladder cancer cells for gene therapy.

The ultimate goal of gene delivery vectors is to establish specific and effective treatments for human diseases. We previously demonstrated that human JC polyomavirus (JCPyV) virus-like particles (VLPs) can package and deliver exogenous DNA into susceptible cells for gene expression. For tissue-specific targeting in this study, JCPyV VLPs were conjugated with a specific peptide for bladder cancer (SPB) that specifically binds to bladder cancer cells. The suicide gene thymidine kinase was packaged and delivered by SPB-conjugated VLPs (VLP-SPBs). Expression of the suicide gene was detected only in human bladder cancer cells and not in lung cancer or neuroblastoma cells susceptible to JCPyV VLP infection in vitro and in vivo, demonstrating the target specificity of VLP-SPBs. The gene transduction efficiency of VLP-SPBs was approximately 100 times greater than that of VLPs without the conjugated peptide. JCPyV VLPs can be specifically guided to target particular cell types when tagged with a ligand molecule that binds to a cell surface marker, thereby improving gene therapy.

Gene therapy for castration-resistant prostate cancer cells using JC polyomavirus-like particles packaged with a PSA promoter driven-suicide gene.

Prostate cancer is the second most common cancer in men globally. Prostate cancer patients at advanced stages are usually treated with androgen deprivation therapy (ADT). However, with disease progression, it often becomes the incurable castration-resistant prostate cancer (CRPC). JC polyomavirus (JCPyV) is a human DNA virus. Its virus-like particles (VLPs) exhibit similar tropism to native virions and they are capable of delivering exogenous genes to the target cells for expression. JCPyV has been detected in prostate cells; therefore, prostate cancer cells may be susceptible to JCPyV infection and JCPyV VLPs may be used as a vector for gene therapy against prostate cancer. Here we constructed a plasmid (pPSAtk) that allows expression of the thymidine kinase suicide gene only in androgen receptor (AR) positive prostate cancer cells using the prostate-specific antigen (PSA) promoter, and used JCPyV VLPs as a vector to carry pPSAtk (PSAtk-VLPs) for transcriptional targeting in prostate cancer cells. In this study, we found that PSAtk-VLPs could only kill AR-positive CRPC 22Rv1 cells in vitro and inhibit the growth of tumor nodules in the xenograft mouse model. Our results reveal that PSAtk-VLPs could potentially be used as a new option for treating CRPC patients in the future.

Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus Extracts Effectively Inhibit BK Virus and JC Virus Infection of Host Cells.

The human polyomaviruses BK (BKPyV) and JC (JCPyV) are ubiquitous pathogens long associated with severe disease in immunocompromised individuals. BKPyV causes polyomavirus-associated nephropathy and hemorrhagic cystitis, whereas JCPyV is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy. No effective therapies targeting these viruses are currently available. The goal of this study was to identify Chinese medicinal herbs with antiviral activity against BKPyV and JCPyV. We screened extracts of Chinese medicinal herbs for the ability to inhibit hemagglutination by BKPyV and JCPyV virus-like particles (VLPs) and the ability to inhibit BKPyV and JCPyV binding and infection of host cells. Two of the 40 herbal extracts screened, Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus, had hemagglutination inhibition activity on BKPyV and JCPyV VLPs and further inhibited infection of the cells by BKPyV and JCPyV, as evidenced by reduced expression of viral proteins in BKPyV-infected and JCPyV-infected cells after treatment with Rhodiolae Kirliowii Radix et Rhizoma or Crataegus pinnatifida Fructus extract. The results in this work show that both Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus may be sources of potential antiviral compounds for treating BKPyV and JCPyV infections.

Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

Inhibition of BK virus replication in human kidney cells by BK virus large tumor antigen-specific shRNA delivered by JC virus-like particles.

Polyomavirus-associated nephropathy (PVAN) due to lytic infection by the BK polyomavirus (BKPyV) remains an important cause of allograft dysfunction and graft loss in renal transplant recipients. PVAN is commonly treated by reducing the dosage of immunosuppressive drugs and adding adjuvant antiviral agents, but the outcomes have been less than satisfactory. The BKPyV early protein large tumor antigen (LT) is indispensable for viral genome replication and viral late protein expression. Therefore, suppressing LT expression may be a way to inhibit BKPyV replication without harming the host human kidney cells. Previous studies have shown that JC polyomavirus (JCPyV) virus-like particles (VLPs), which have tropism for the human kidney, can package and transfer exogenous genes into human kidney cells for expression. In this study, we constructed an expression plasmid for a BKPyV LT-specific shRNA (shLT) and used JCPyV VLPs as a delivery vehicle to transduce the shLT plasmid into BKPyV-infected human kidney cells. The expression of BKPyV early (LT) and late (VP1) proteins was examined after transduction by immunofluorescence microscopy and Western blotting. We found that transduction with the shLT plasmid decreased the proportions of BKPyV LT- and VP1-expressing cells by 73% and 82%, respectively, relative to control. The viral genomes were also decreased by 56%. These results point to the promising possibility of developing shLT-transducing JCPyV VLPs as a specific anti-BKPyV approach for PVAN treatment.