Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors.
ABSTRACT: Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.
Project description:Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.
Project description:Gene therapy for hematopoietic diseases has been hampered by the low frequency of transduction of human hematopoietic stem cells (HSCs) with retroviral vectors pseudotyped with amphotropic envelopes. We hypothesized that transduction could be increased by the use of retroviral vectors pseudotyped with envelopes that recognize more abundant cellular receptors. The levels of mRNA encoding the receptors of the feline retroviruses, RD114 and feline leukemia virus type C (FeLV-C), were significantly higher than the level of gibbon ape leukemia virus (GaLV) receptor mRNA in cells enriched for human HSCs (Lin- CD34+ CD38-). We cotransduced human peripheral blood CD34+ cells with equivalent numbers of FeLV-C and GALV or RD114 and GALV-pseudotyped retroviruses for injection into fetal sheep. Analysis of DNA from peripheral blood and bone marrow from recipient sheep demonstrated that FeLV-C- or RD114-pseudotyped vectors were present at significantly higher levels than GALV-pseudotyped vectors. Analysis of individual myeloid colonies demonstrated that retrovirus vectors with FeLV-C and RD114 pseudotypes were present at 1.5 to 1.6 copies per cell and were preferentially integrated near known genes We conclude that the more efficient transduction of human HSCs with either FeLV-C- or RD114-pseudotyped retroviral particles may improve gene transfer in human clinical trials.
Project description:The amphotropic murine retrovirus receptor Ram-1 shows significant sequence similarity to the gibbon ape leukemia virus (GALV) receptor Glvr-1, and both of these cell surface virus receptors normally function as sodium-dependent phosphate symporters. However, Ram-1 from humans or rats does not serve as a receptor for GALV, and Glvr-1 from humans does not serve as a receptor for amphotropic virus. Here we show that the murine retrovirus 10A1 can enter cells by using either Glvr-1 or Ram-1. Furthermore, we have constructed Ram-1/Glvr-1 hybrid receptors that allow entry of both GALV and amphotropic virus. While GALV and amphotropic virus are in separate interference groups when assayed on human cells, they do interfere with each other in cells expressing the hybrid receptor. These results indicate a close functional relationship between retroviruses that utilize members of this newly defined receptor family and provide a molecular explanation for nonreciprocal and cell type-specific interference observed for some retrovirus classes.
Project description:Identification and cloning of the receptors for amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV) have both enabled the determination of the normal function of these virus receptors in cells and initiated experimental examination of how these receptors interact with their respective viruses. GaLV and A-MuLV have distinct host ranges and use different receptors to infect human cells. It was therefore surprising to find that the human GaLV and A-MuLV receptors were not only structurally similar but performed similar cellular functions (B. O'Hara, S. V. Johann, H. P. Klinger, D. G. Blair, H. Rubinson, K. J. Dunn, P. Sass, S. M. Vitek, and T. Robbins, Cell Growth Differ. 1:119-127, 1990; M. van Zeijl, S. V. Johann, E. Closs, J. Cunningham, R. Eddy, T. B. Shows, and B. O'Hara, Proc. Natl. Acad. Sci. USA 91:1168-1172, 1994; M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994; and Z. Olah, C. Lehel, W. B. Anderson, M. V. Eiden, and C. A. Wilson, J. Biol. Chem., in press). We have now determined that the murine retrovirus 10A1 can use both the human GaLV receptor and the human A-MuLV receptor to infect cells. Furthermore, we have cloned and functionally characterized a unique form of the amphotropic receptor homolog expressed in E36 hamster cells. This receptor (EAR) can serve as both a GaLV receptor and an A-MuLV receptor, and it therefore differs from the receptors expressed in human cells, which function exclusively as either GaLV or A-MuLV receptors.
Project description:We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.
Project description:Both cell-free and cell-associated infection routes are important for retroviral dissemination. Regardless of the mechanism, the driving force of retroviral entry is the interaction between the viral envelope and its receptor. To date it remains unclear how decreased affinity of viruses for their receptors affects viral cell-free infection, cell-cell transmission, and spreading kinetics. We have previously characterized a mutant form of the amphotropic murine retrovirus receptor human phosphate transporter 2 (PiT2) wherein the single substitution of a glutamic acid for the lysine residue at position 522 of this receptor is sufficient to render it to function as a gibbon ape leukemia virus (GALV) receptor.In this study we analyzed the binding affinity of the mutant receptor PiT2K522E and determined that it has a 1000 fold decreased GALV envelope binding affinity compared to the GALV wild type receptor. The decreased affinity does not restrict the initiation of cell-free GALV infection. The diminished binding affinity does, however, correlate with a decrease in the ability of GALV to spread in cells expressing this mutant receptor.The reduced ability of GALV to subsequently spread among cells expressing PiT2K522E is likely resulted from reduced cell-cell transmission, the decreased ability of PiT2K522E-expressing cells to establish superinfection interference, and attendant cytopathic affects.
Project description:10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.
Project description:Glvr1 encodes the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related gene Glvr2 encodes the human receptor for amphotropic murine leukemia viruses (A-MLVs). The two proteins are 62% identical in their amino acid sequences and are predicted to have 10 transmembrane domains and five extracellular loops. A stretch of nine amino acids (region A) in the predicted fourth extracellular loop was previously shown to be critical for the function of Glvr1 as receptor for GALV and FeLV-B. Glvr1 and -2 show clusters of amino acid differences in several of their predicted extracellular loops, with the highest degree of divergence in region A. Chimeras were made between the two genes to further investigate the role of Glvr1 region A in defining receptor specificity for GALV and FeLV-B and to map which regions of Glvr2 control receptor specificity for A-MLVs. Region A from Glvr1 was sufficient to confer receptor specificity for GALV upon Glvr2, with the same chimera failing to act as a receptor for FeLV-B. However, introduction of additional N- or C-terminal Glvr1-encoding sequences in addition to Glvr1 region A-encoding sequences resulted in functional FeLV-B receptors. Therefore, FeLV-B is dependent on Glvr1 sequences outside region A for infectivity. The receptor specificity of Glvr2 for A-MLV could not be mapped to a single critical region; rather, N-terminal as well as C-terminal Glvr2-encoding sequences could confer specificity for A-MLV infection upon Glvr1. Surprisingly, though GALV/FeLV-B and A-MLV belong to different interference groups, some chimeras functioned as receptors for all three viruses.
Project description:The Chinese hamster cell lines E36 and CHOK1 dramatically differ in susceptibility to amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GALV); E36 cells are highly susceptible to both viruses, CHOK1 cells are not. We have previously shown that GALV can infect E36 cells by using both its own receptor, HaPit1, and the A-MuLV receptor, HaPit2. Given that the two cell lines are from the same species, the loss of function of both of these receptors in CHOK1 cells is surprising. Other studies have shown that CHOK1 cells secrete proteins that block A-MuLV entry into CHOK1 as well as E36, suggesting the two A-MuLV receptors are functionally identical. However, CHOK1 conditioned medium does not block GALV entry into E36, indicating the secreted inhibitors do not block HaPit1. HaPit1 and ChoPit1 therefore differ as receptors for GALV; ChoPit1 is either inactivated by secreted factors or intrinsically nonfunctional. To determine why GALV cannot infect CHOK1, we cloned and sequenced ChoPit1 and ChoPit2. ChoPit2 is almost identical to HaPit2, which explains why CHOK1 conditioned medium blocks A-MuLV entry via both receptors. Although ChoPit1 and HaPit1 are 91% identical, a notable difference is at position 550 in the fourth extracellular region, shown by several studies to be crucial for GALV infection. Pit1 and HaPit1 have aspartate at 550, whereas ChoPit1 has threonine at this position. We assessed the significance of this difference for GALV infection by replacing the aspartate 550 in Pit1 with threonine. This single substitution rendered Pit1 nonfunctional for GALV and suggests that threonine at 550 inactivates ChoPit1 as a GALV receptor. Whether native ChoPit1 functions for GALV was determined by interference assays using Lec8, a glycosylation-deficient derivative of CHOK1 that is susceptible to both viruses and that has the same receptors as CHOK1. Unlike with E36, GALV and A-MuLV exhibited reciprocal interference when infecting Lec8, suggesting that they use the same receptor. We conclude both viruses can use ChoPit2 in the absence of the inhibitors secreted by CHOK1 and ChoPit1 is nonfunctional.
Project description:Chinese hamster ovary (CHO) cells are resistant to infections by gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MLV) unless they are pretreated with tunicamycin, an inhibitor of N-linked glycosylation. These viruses use the related sodium-phosphate symporters Pit1 and Pit2, respectively, as receptors in nonhamster cells, and evidence has suggested that the corresponding transporters of CHO cells may be masked by tunicamycin-sensitive secreted inhibitors. Although the E36 line of Chinese hamster cells was reported to secrete the putative Pit2 inhibitor and to be sensitive to the inhibitory CHO factors, E36 cells are highly susceptible to both GALV and A-MLV in the absence of tunicamycin. Moreover, expression of E36 Pit2 in CHO cells conferred tunicamycin-independent susceptibilities to both viruses. Based on the latter results, it was suggested that E36 Pit2 must functionally differ from the endogenous Pit2 of CHO cells. To test these ideas, we analyzed the receptor properties of CHO Pit1 and Pit2 in CHO cells. Surprisingly, and counterintuitively, transfection of a CHO Pit2 expression vector into CHO cells conferred strong susceptibility to both GALV and A-MLV, and similar overexpression of CHO Pit1 conferred susceptibility to GALV. Thus, CHO Pit2 is a promiscuous functional receptor for both viruses, and CHO Pit1 is a functional receptor for GALV. Similarly, we found that the natural resistance of Mus dunni tail fibroblasts to subgroup C feline leukemia viruses (FeLV-C) was eliminated simply by overexpression of the endogenous FeLV-C receptor homologue. These results demonstrate a novel and simple method to unmask latent retroviral receptor activities that occur in some cells. Specifically, resistances to retroviruses that are caused by subthreshold levels of receptor expression or by stoichiometrically limited masking or interference mechanisms can be efficiently overcome simply by overexpressing the endogenous receptors in the same cells.