Murine leukemia virus (MuLV)-based retroviral replicating vectors (RRVs) have been developed for cancer gene therapy due to their tumor selectivity, high transduction efficiency, and low viral clearance (1, 2). However, there are limitations in the size of therapeutic genes that can be delivered using these vectors due to the highly compact nature of the MuLV genome (3, 4). The size of the therapeutic gene correlates with increased deletion of the introduced sequence, resulting in attenuated kinetics (3). Therefore, only relatively small genes such as the cytosine deaminase (CD) gene can be used for cancer gene therapy studies with the RRV system (5, 6).
To overcome the packaging size limitation, the sRRV system was developed (7, 8). In this system, the
MuLV envelope glycoprotein interacts with Pit2 and infects both rodent and human cells, while GaLV infects heterologous cells but not rodent cells through an interaction with Pit1 (9-13). Host cells can therefore be co-infected with MuLV and GaLV without superinfection interference (14). To improve the infectivity of retroviruses in diverse cells, recombinant chimeric retroviruses have been engineered in which the envelope protein of the amphotropic virus is replaced with that of another virus type. It has been shown that GaLV-pseudotyped recombinant retroviruses produced high-titer viral vectors (15-17) and also showed improved gene transfer efficiency in human progenitor cells and in baboons (18-20).
In the present study, we constructed MuLV-based, GaLV-pseudotyped semi-retroviral vectors (spRRV system) with improved transduction efficiency. The spRRV vectors propagated more quickly than sRRV system (MuLV-based semi-retroviral vectors)
We developed a semi-MuLV-based, GaLV-pseudotyped RRV (spRRV). This spRRV system is composed of two complementary replication-defective vectors, MuLV-Gag-Pol (sRRVgp) and GALV-Env (spRRVe), which express the
To determine whether semi-RRVs were capable of replicating efficiently in cultured cells, human glioblastoma U-87 MG cells were transduced with the same number of genomic copies (gc) of the sRRV or spRRV. sRRVgp-RFP + sRRVe-GFP (sRRV-FL) and sRRVgp-RFP + spRRVe-GFP (spRRV-FL) were used to examine the spread efficiency of each gene delivery system. At 20 days post-infection, the percentage of GFP-expressing spRRV-FL-transduced U-87 MG cells was nearly 100%, but only approximately 30% of sRRV-FL-transduced cells expressed GFP (Fig. 2A, B). We, as expected, observed the superinfection resistance phenomenon showing a plateau state in the proportion of RFP-positive cells at a low ratio as compared to GFP-positive cells (Fig. 2B). As with the experimental results for cell lines, both the GFP and RFP signals were strong and restricted to the tumor area in spRRV-FL-injected mice, whereas the GFP and RFP signals in sRRV-FL-injected tumors were weak (Fig. 2C). Tumor cells from sRRV-FL injected mice showed relatively modest transduction efficiency and 53.433 ± 7.4% of cells were GFP-positive. On the other hand, the percentage of cells expressing GFP in spRRV-FL-injected animals was 84 ± 1.48% (Fig. 2D), 1.5-fold higher than that of sRRV-FL (P = 0.0046). These data indicate that the transduction efficiency of spRRV-FL is significantly improved compared to sRRV-FL.
We next evaluated the level of the HSV1-
The anti-tumor activity of
Here, we report the construction of a pseudotyped, semi-replicating retroviral vector that largely enhanced the transduction efficiency of sRRV and thereby prolonged the survival of the glioblastoma model mouse. To improve the transduction efficiency of the retroviral vector, the previously reported sRRV was modified in two ways. First, one of the two retroviral vectors was altered to encode the GaLV envelope glycoprotein instead of the MuLV envelope glycoprotein. Second, the internal promoter (mCMV MIEP) for the therapeutic gene expression was inserted instead of IRES sequences. Splitting the RRV viral genome into two vectors and pseudotyping one viral vector with GaLV envelope glycoprotein would provide several advantages as compared with conventional RRV vectors. First, it can deliver multiple therapeutic genes at the same time into the target organs. Tumor tissue is a collection of heterogeneous cells with differences in cell markers, genetic abnormality, growth rate, and apoptosis (23). These characteristics of tumor tissue make it hard to treat cancer because some cells would escape from certain kinds of therapy and provide resistance. Delivery of multiple therapeutic genes at the same time would provide advantages on tumor treatment through the synergistic effect of each therapeutic gene (24, 25). Second, the relatively small size of the retroviral packaging vector enables to encode of large size of the therapeutic gene. Despite the efficient propagation of viral vectors with a stable viral genome, conventional RRVs bear limitations in the size of the therapeutic gene because of the compact nature of the MuLV genome. Only less than 1.2 Kb transgene including the internal promoter can be encoded in RRVs because the larger size of the therapeutic gene would reduce the propagation of the virus. In the case of the spRRV system, however, therapeutic genes of approximately 3 kb and 4.5 kb including a promoter are able to be inserted both in our sRRVgp and spRRVe vectors, respectively.
As in the
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Korea Food and Drug Administration. All animal studies were reviewed and approved by the Animal Care and Use Committee of the Chungnam National University Animal Resource Center (permit number CNU-00822).
U87-MG and 293T cells were obtained from American Type Culture Collection (ATCC, Bethesda, MD, USA) where cell line authentication and species identification were performed. Cells were grown in Dulbecco’s modified Eagle medium with 10% fetal bovine serum and antibiotics. All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.
The sRRV vectors (spRRV-FL, sRRV-FL, sRRV-TK and spRRV-TK) were produced by transient transfection of 293T-cells with the sRRV plasmid vectors using Lipofectamine Plus. After 48 h, the supernatant was harvested, filtered through 0.2 μm syringe filters, and stored at −80°C. Real-time PCR was performed with a Retrovirus Titer Set (TAKARA) to determine viral titers.
Replication kinetics were analyzed
To assess HSV-TK gene expression in RRV-infected cells, U-87 MG were transduced with 1 × 107 gc of sRRV-TK, spRRV-TK, or sRRV-FL. Cells were harvested 7 days after infection and were resuspended in lysis buffer composed of 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), and a protease inhibitor cocktail (Gen Depot). Each sample was boiled in SDS loading dye and resolved by 10% SDS-PAGE. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Immunoblotting was performed with a goat polyclonal anti-HSV-TK primary antibody for 2 h at room temperature (1:1000, Santa Cruz Biotechnology) and a peroxidase-conjugated donkey anti-goat secondary antibody for 1 h at room temperature (1: 5000, Santa Cruz Biotechnology). Bound antibodies were detected using the ECL Plus system (GE Healthcare Life Sciences, Amersham, UK). An α-tubulin antibody was used as an internal loading control (1:1000, Santa Cruz Biotechnology).
The toxicity of GCV (Sigma) was determined
spRRV-TK was administered intratumorally at 4.5 × 107 gc 7 days after injection of U-87 MG cells into the mouse brain. On days 4, 7, 10, 14, and 17, animals were injected with 18.5 MBq 18F-FHBG via the tail vein. An hour after substrate uptake, 18F-FHBG PET-CT scans were performed in three-dimensional (3-D) acquisition mode (eXplore VistaCT, GE). For CT scans, the X-ray conditions were 250 μA and 40 kV for 6 min. The CT resolution was 200 μm, and the number of acquired projections was 360. For PET scans, normalization was applied, while scatter corrections and attenuation corrections were not. PET scans were acquired for 10 min per bed position for all studies. All images were reconstructed with iterative reconstruction (OSEM 2-D, 32 subsets, two interactions). Images were normalized as standardized uptake values (SUV) using the formula: SUV = decay-corrected mean tissue activity concentration (in Bq/ml)/[injected dose (in Bq) × body weight (in g)]. MR images were obtained by an animal MRI system (7 T Biospin, Bruker). Animal PET-CT and MRI systems shared one bed for PET-MR image fusion. All PET/CT/MR data analyses, including multimodal images and 3-D images, were compiled using OsiriX imaging software (www.osirix-viewer.com).
Intracerebral U-87 MG glioblastomas were established as described above (n = 34). One week after the tumor inoculation, the animals were stereotaxically injected with spRRV-FL or spRRV-TK (4.5 × 107 gc/10 μl). Two weeks after the vector inoculation, the spRRV-TK-injected animals were split into two groups. One group (n = 15) received daily intraperitoneal injections of GCV (100 mg/kg) for 30 days, while the other group (n = 10) received daily intraperitoneal injections of PBS for 30 days. spRRV-FL-injected mice (n = 9) received daily intraperitoneal injection of GCV (100 mg/kg) for 30 days. Experimental mice that survived for 150 days and control mice that survived for more than 30 days after tumor establishment were perfused. The brains were then dissected and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 5 μm and the sections were stained with H&E.
Student’s t-tests were used for the cell viability and FACS experiments. Survival percentages were assessed using Kaplan-Meier survival analyses. Prism 5 statistical software was used for all analyses (GraphPad Software). P-values that were < 0.05 were considered to be statistically significant.
This research was funded by grants from the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIT) (NRF-2018M3A9B5059544).
The authors have no conflicting interests.