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.


GCV
Ganciclovir SPB Specific peptide for bladder cancer TAMRA Tetramethylrhodamine tk Thymidine kinase VLP Virus-like particle Gene therapy involves the delivery of therapeutic genes into the patient's body to treat disease as an alternative to traditional treatments [1][2][3] . Since the first gene therapy trial was approved in 1989 4 , nearly 2600 gene therapy trials have been completed or are underway worldwide 5 . The majority of these clinical trials involve treatments for cancer (65.0%), along with treatments for inherited monogenic diseases (11.1%) and infectious diseases (7.0%). However, safely protecting the entry of genes into specific target cells without harming nontarget cells remains the greatest challenge for gene therapy. During the process of evolution, viruses developed a mechanism that specifically protects the entry of viral genes into susceptible cells. Viral vectors derived from viruses retain this property, thus providing an attractive option as gene therapy vectors [6][7][8] . We previously reported the first cloning and expression of the major capsid protein VP1 of JC polyomavirus (JCPyV) in insect cells 9 , Escherichia coli 10 , and yeast 11 . The recombinant VP1 protein is capable of self-assembly into a virus-like particle (VLP) for gene delivery. As a gene delivery vector, recombinant JCPyV VLPs can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLPs without requiring viral genetic material, which can be subsequently delivered into tissues susceptible to JCPyV to enable gene transduction and gene therapy. These VLPs were successfully used to package and deliver exogenous DNA into human kidney cells for expression 9 . We also used these VLPs to deliver an antisense oligodeoxynucleotide www.nature.com/scientificreports/ into human glioma (SVG) cells, which resulted in growth inhibition in these cells 12 . More recently, we demonstrated the ability of these VLPs to deliver a suicide gene, thymidine kinase (tk), into human colon carcinoma cells 13 , diffuse large B-cell lymphoma cells 14 , and glioblastoma cells 15 . For tissue-specific gene expression, tissuespecific promoters were constructed to drive the expression of the suicide gene, which was delivered by the VLPs to inhibit the growth of bladder cancer cells 16 , lung adenocarcinoma cells 17,18 , and prostate cancer cells 19 in a mouse model. Furthermore, the VLPs were used to successfully deliver a BK polyomavirus (BKPyV) LT peptidespecific small hairpin RNA to inhibit BKPyV replication in human kidney cells 20 and to deliver an interleukin (IL)-10 RNA interference vector into macrophage cells to reduce IL-10 expression 21 as a possible gene therapy for systemic lupus erythematosus. Moreover, we found that the VLPs could package exogenous DNA molecules of up to 9.4 kb in length 22 . The development of a gene delivery vector using JCPyV VLPs has been reviewed previously 23 . In this study, we extend this development in a further attempt to conjugate these VLPs with a bladder cancer-binding peptide to specifically target bladder cancer cells. The BC, DE, and HI loops are the exposed domains in the JCPyV VP1 molecule that interact with cell receptors. Therefore, polymorphisms in these regions affect the binding of the virus to host cell receptors and may be associated with viral toxicity or progressive multifocal leukoencephalopathy (PML) development and severity [24][25][26] . Accordingly, altering or modifying the exposed domains of JCPyV VLPs can change their initial binding ability or affinity for host cell receptors. In recent years, phage display technology has been used to find cancer-specific peptides, which have successfully been used to deliver therapeutic genes or drugs to tumors, thereby specifically killing tumor cells 27 . A specific peptide for bladder cancer (abbreviated SPB; peptide sequence: CSNRDARRC) was discovered by Lee et al. 28 and can be used for the diagnosis or treatment of bladder cancer. In this study, this SPB was conjugated with JCPyV VLPs for the investigation of bladder cancer-specific targeting.
Urothelial carcinoma bladder cancer is the second most common cancer of the urinary tract. Approximately 75% of bladder cancers are non-muscle-invasive bladder cancers (NMIBCs), which are characterized by a high recurrence rate. Approximately 25% of patients have muscle-invasive bladder cancer (MIBC) and must undergo radical cystectomy 29,30 . Thus, patients with NMIBC require regular lifelong invasive cystoscopies to monitor tumor recurrence or progression. In the present study, we used sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) to chemically link JCPyV VLPs and a SPB to form JCPyV VLP-SPBs. We further tested whether JCPyV VLP-SPBs packaging a suicide tk gene can specifically target human bladder cancer cells and inhibit the growth of these cells in vitro and in a mouse xenograft model in vivo.

Results
SPB binding specificity. Previously, a human bladder cancer cell-specific binding peptide (i.e., SPB) was discovered by Lee et al. 28 . Here, we first confirmed the binding specificity of this SPB to bladder cancer cells. The SPB was linked to the fluorescent dye tetramethylrhodamine (TAMRA) to form a TAMRA-SPB conjugate. Fluorescence microscopy showed that the red fluorescent SPB (TAMRA-SPB) bound specifically to HT-1376 bladder cancer cells (Fig. 1). As the TAMRA-SPB concentration increased, the red fluorescence of the HT-1376 cells exhibited a concentration-dependent effect (Fig. 1). To further confirm the specificity of TAMRA-SPB binding to HT-1376 cells, we used unconjugated SPB to compete with TAMRA-SPB binding. With increasing concentrations of unconjugated SPB, the red fluorescence of TAMRA-SPB decreased (Fig. 2). This result demonstrated that this SPB can be used to achieve specific binding to bladder cancer cells.
Binding specificity of JCPyV VLP-SPBs to bladder cancer cells. To increase the specificity of gene targeting, we attempted to link the SPB peptide and JCPyV VLPs by (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (sulfo-SMCC) crosslinker. Compared to the X-ray diffraction pattern simulating SV40 VP1 (PDB: 1SVA) 31 , nine amino acids with primary amines are located in outer loops of JCPyV VP1 molecule, allowing stable covalent conjugation with SPB peptide by sulfo-SMCC. We next tested the cell binding specificity of JCPyV VLP-SPBs to bladder cancer cells in comparison to human lung cancer cells (A549) and neuroblastoma cells (IMR-32). JCPyV VLPs conjugated with TAMRA-SPB, namely, JCPyV VLP-SPB-TAMRA, were used to determine the binding specificity of JCPyV VLP-SPBs to bladder cancer cells. When 1 µg, 3 µg, or 10 µg of JCPyV VLP-SPB-TAMRA was separately added to A549 and IMR-32 cells, no red fluorescence representing JCPyV VLP-SPB-TAMRA was observed (Fig. 3A,B). In contrast, slight red fluorescence representing JCPyV VLP-SPB-TAMRA was detected after addition to HT-1197 (Fig. 3C) and HT-1376 (Fig. 3D) bladder cancer cells at low concentrations (1 µg, 3 µg), and obvious red fluorescence was observed in both cell lines when the concentration of JCPyV VLP-SPB-TAMRA was increased to 10 µg (Fig. 3C,D). In our previous study, we have demonstrated that JCPyV VLP can successfully deliver exogenous DNA into lung cancer cells (A549) 17,18 , neuroblastoma cells (IMR-32) 12 , and bladder cancer cells (HT-1197) 16 for gene expressions. After conjugated with bladder cancer specific peptide SPB, JCPyV VLP specifically binds to bladder cancer cells, simultaneously losing its binding to susceptible lung and neuroblastoma cells. This result demonstrates that conjugation with a specific peptide allows alteration of the original tropism of JCPyV VLPs.

Specific inhibition of bladder cancer cell growth by tk-JCPyV VLP-SPBs in a xenograft mouse model.
After testing the specific cytotoxicity of tk-JCPyV VLP-SPBs in bladder cancer cells, we further tested the specificity of tk-JCPyV VLP-SPB cytotoxicity to bladder cancer cells in an animal model. As shown in Fig. 4, linking a specific peptide of bladder cancer cells has changed the tropism of JCPyV VLP to lung cancer cells. We proposed that after conjugated with specific peptide of bladder cancer cells, tk-JCPyV VLP-SPBs will specifically deliver the packaged tk gene into bladder tumor nodules instead of lung tumor nodules. We subcutaneously injected A549 lung cancer cells and HT-1376 bladder cancer cells into the left and right sides, respectively, of the same mouse. One week later, the mice were randomly divided into five groups, 3 Fig. 5A,B). The body weight of the mice showed no significant difference during the experiment (data not shown). These results further demonstrated that the specific targeting of JCPyV VLPs can be altered through conjugation to a specific peptide in an in vivo animal model through the tail vein administration.

Discussion
In the present study, we conjugated a bladder cancer-specific peptide (i.e., SPB) with JCPyV VLPs and confirmed that the resulting JCPyV VLP-SPBs bound specifically to bladder cancer cells. JCPyV VLP-SPBs effectively delivered the tk suicide gene, which is cytotoxic to bladder cancer cells, only to bladder cancer cell lines and not to cell lines originally demonstrated to be susceptible to JCPyV VLPs, such as lung cancer and neuroblastoma cell lines. The xenograft mouse model also showed that JCPyV VLP-SPB only inhibited the growth of bladder tumors, while the growth of lung cancer cells on the contralateral side was unaffected. Therefore, the present study demonstrated that conjugation with a specific peptide can redirect JCPyV VLPs to specific tissues and can also significantly increase the transduction specificity and efficiency of JCPyV VLPs as a gene therapy vector. VLPs have no viral genes but retain the characteristics of virus-specific binding to host cells, thus functioning as an important type of gene delivery vector in the development of gene therapy 32 . However, the ease of preparation and the cost of gene delivery vectors are also important factors to consider in vector development. JCPyV VLPs are produced in E. coli and self-assemble into VLPs, which can be easily purified by sucrose gradient centrifugation; thus, they are easy to obtain in a cost-effective manner 10 . During assembly, JCPyV VLPs can package DNA molecules of up to approximately 9.4 kb in size, which is sufficient to address current limitations regarding the size of genes required for treatments 22 . JCPyV VLPs are an efficient gene therapy vector also because they can deliver and protect the entry of genes into both dividing and nondividing cells 23 . To achieve tissue specificity, we previously used JCPyV VLPs to deliver tissue-specific promoters to specifically express a gene of interest in www.nature.com/scientificreports/ a particular cell; these promoters included the lung cancer-specific SPB promoter 17,18 , prostate cancer-specific PSA promoter 19 , and MUC1 promoter, which is overexpressed in bladder cancer 16 . In the current study, we further conjugated JCPyV VLPs with a bladder cancer cell-specific binding peptide for investigation of specific targeting. This conjugation allowed JCPyV VLPs to target bladder cancer cells and subsequently inhibited their growth, demonstrating that conjugation with a specific peptide increased the therapeutic specificity of JCPyV VLPs. In addition, the results also show a 100-fold increase in efficiency in terms of tumor weight in terms of therapeutic efficiency. Therefore, JCPyV VLPs can be guided by a cell surface marker binding peptide for the specific delivery and transduction of a gene of interest expressed by a tissue-specific promoter in a targeted cell type. VLPs retain the characteristics of virus-specific binding to host cells, but are allowed for genetic or chemical modification to alter the tropism or increase their binding efficiency to their nature host cells 33,34 . For example, the specific cellular targeting of MS2 VLPs are demonstrated through attachment of a tissue specific nucleic acid aptamers 35 . Conjugation of folic acid on VLP derived from truncated hepatitis B virus core antigen (tHBcAg) increases the specificity and efficacy of the drug delivery 36 . By genetic incorporation of cancer specific peptides to the hybrid adeno-associated virus/phage (AAVP) particles results in an increase gene transfer efficiency 37 . In the current study, we demonstrated that the tropism of JCPyV VLP was altered after conjugated with the specific peptide for bladder cancer, SPB. Among the types of bladder cancers, urothelial carcinoma is characterized by high recurrence, and approximately 10-20% of cases progress to MIBC and metastasis. Many drugs have been developed for the treatment of metastatic bladder cancer, but the clinical response remains limited 29 . The HSV-tk/GCV system is one of the most commonly used and studied prodrug systems. Viral thymidine kinases convert guanosine analog GCV into GCV monophosphate which can be easily converted into toxic triphosphate GCV by cellular kinases. When triphosphate GCV is incorporated into replicating DNA, a termination of DNA replication and cell apoptosis will occur. The human cellular kinases exhibit much lower affinity to convert GCV compared to HSV-tk. Only cells receiving HSV-tk gene can convert GCV (prodrug) to monophosphate GCV, and ultimately toxic triphosphate GCV (drug) 38,39 . In addition, a bystander effect which can amplify the toxicity of triphosphate GCV is also www.nature.com/scientificreports/ observed in HSV-tk/GCV system, which makes HSV-tk/GCV system an attractive strategy for cancer therapy 39,40 . However, the clinical trial of HSV-tk/GCV system did not show promising effect for patients with glioblastoma multiforme and advanced hepatocellular carcinoma 41,42 . The gene transfer efficiency is one of the challenges in these systems. In the present study, HSV-tk gene was packaged in JCPyV VLPs and conjugated with specific bladder cancer peptide, SPB. The tk-JCPyV VLP-SPBs were administered to mice via the tail vein and effectively inhibited the growth of bladder tumor nodules in the mice. These results demonstrated that after tk-JCPyV VLP-SPB entered the circulation; it specifically targeted bladder cancer cells. The findings also suggested that JCPyV VLPs can be applied through i.v. injections for the treatment of metastatic tumors if conjugated with a tissuespecific ligand. However, delivery of a large quantity of JCPyV VLP-SPB may also have off-target effects, as shown in cytotoxicity tests in Fig. 3A,B. Furthermore, most people are seropositive to JCPyV, the use of JCPyV VLPs as a delivery vector may result in a decreased efficiency. In order to avoid the immune elimination, a modification of the surface of JCPyV VLPs may be a strategy for developing the JCPyV VLPs as a human gene delivery vector.
In summary, JCPyV VLPs can function as a gene therapy vector and can package a gene of interest driven by a tissue-specific promoter. The present results further demonstrated that conjugation with specific peptides allows the original tropism of JCPyV VLPs to be altered, thereby redirecting the encapsidated genes for expression in specific tissues and enhancing the gene transduction efficiency. Therefore, JCPyV VLPs could be designed as a flexible gene delivery vector targeted to different cell types for a variety of therapeutic purposes.

Methods
Cell lines. All cell lines were purchased from the Bioresource Collection and Research Center, Taiwan. The culture medium for the human bladder cancer cell lines HT-1197 and HT-1376 was minimum essential medium supplemented with l-glutamine, nonessential amino acids, sodium pyruvate, fetal bovine serum, and penicillin/streptomycin antibiotic solution (Thermo Fisher Scientific, Cambridge, MA, USA). Human lung adenocarcinoma (A549) and neuroblastoma (IMR-32) cell lines were cultured in culture dishes (Guangzhou Jet Biofiltration Co. Ltd. Guangzhou, China) with Dulbecco's modified Eagle medium supplemented with nonessential amino acids, sodium pyruvate, fetal bovine serum, and penicillin/streptomycin antibiotic solution (Thermo).

SPB binding to bladder cancer cells.
The SPB peptide (CSNRDARRC), which has specificity for bladder cancer 28 , was synthesized by AngeneBiotech (Taipei, Taiwan). The red fluorescent tetramethylrhodamine (TAMRA) molecules were simultaneously added to the N-terminus of SPB in the process of synthesis (Angen-eBiotech), which is named as TAMRA-SPB. To test the binding of TAMRA-SPB to HT-1376 cells, 5 × 10 5 cells were seeding onto a coverslip (Marienfeld, Lauda-Königshofen, Germany) placed at 4 °C and allowed to stand for 10 min. The culture medium was removed, and the cells were washed twice with cold PBS. After the addition of 20 µM, 50 µM, or 100 µM TAMRA-SPB, the mixture was placed at 4 °C for 30 min, and the cells were washed three times with 4 °C PBS. The cells were then mounted using a mounting solution containing 0.1% DAPI (Sigma-Aldrich, D9542) and observed under a confocal microscope (Olympus, FluoView 1200) with 400X magnification.
Testing the specificity of the SPB for bladder cancer cells. 5 × 10 5 cells of HT-1376 cells were placed at 4 °C and allowed to stand for 10 min. The culture medium was removed, and the cells were washed twice with cold PBS. After the addition of 0.002, 0.02, 0.2, 2, 20, or 200 μM SPB to TAMRA-SPB (20 μM), HT-1376 cells were added to the mixture, placed at 4 °C for 30 min, and washed three times with 4 °C PBS. Cells were mounted using a mounting solution containing 0.1% DAPI and observed using a confocal microscope (Olympus, FluoView 1200) with 400X magnification.