Cancer Gene Therapy – Suicide gene therapy of sarcoma cell lines using recombinant adeno-associated virus
Gene delivery systems that are currently being evaluated by clinical trials include retroviruses, lentiviruses, foamiviruses, adeno and adenoassociated viruses, herpesviruses and cationic liposomes/DNA–protein complexes. It is associated with resistance to conventional anticancer treatments. They are rare neoplasms, comprising up to 1% of all malignant tumors of adults. This highlights the growing need to develop antitumor therapies, which target hypoxic cells and which might, therefore, be used in combination with existing modalities of cancer therapy to kill both normoxic and hypoxic regions of tumors. At each given time point, the percentages of cleaved caspae-3, caspase-9 and TUNEL positive cells were significantly higher in the ADV.tk + GCV group than saline group (P < 0.005), while CD31 and VEGF staining were significantly less in ADV.tk + GCV group than in saline group (P < 0.005). Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells. When ponicidin at the concentration without antiviral activities (0.2 g/mL) was combined with ACV or GCV, the cytotoxic levels in HSV-TK-expressing cells were enhanced by 3- to 87-fold and 5- to 52-fold, respectively, compared with the nucleoside alone.
We investigated whether increasing the level of expression of gap junctions in glioma cells increased the efficacy of gene therapy or chemotherapy regimens for the treatment of these brain tumors. In the present study, we used the nonviral vector polyethylenimine (PEI), which has been shown to be a promising alternative to viral vectors in applications in vivo and a suitable backbone for coupling to targeting ligands.22, 23, 24 We employed PCI to improve and target PEI-mediated delivery of the HSVtk gene. Ganciclovir (GCV) is a nucleoside analog that is phosphorylated by TK to GCV monophosphate and is further phosphorylated by the cellular kinases to GCV diphosphate and GCV triphosphate. GCV triphosphate competes with deoxyguanosine triphosphate for the DNA polymerase and, as a result, DNA synthesis is impaired by chain termination resulting in cell death in the dividing cells.9 Interestingly, besides the direct and indirect toxicity (bystander effect), an immunotoxic effect has also been observed for the TK/ganciclovir system, the mechanism of which has not been fully elucidated yet.7, 10 We and others have investigated the infectability of tumor cells by recombinant adeno-associated virus 2 (rAAV-2) vectors.11, 12, 13, 14, 15, 16 For further reading on TK-mediated gene therapy, please refer to a recent review by Fillat et al.17 In this study, we investigated the susceptibility of six human sarcoma cell lines to rAAV-2 suicide vectors and the subsequent killing of the infected sarcoma cells following exposure to the prodrug ganciclovir. Although HSV-tk/neor transduction and G418 selection yielded GCV-sensitive T cells, this selection procedure is undesirable because of nonspecific toxicity11, prolonged culture periods6,7,10, immunogenicity of the bacterial neor gene9,12, loss of functional alloreactive T cells11,13, and an inability to efficiently track the gene-modified cells both in vitro and in vivo. coli strain DH5 (F- 80lacZM15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1; Life Technologies, Lofer, Germany) was used. Upon formation of the death-inducing signaling complex in the extrinsic death receptor pathway or the apoptosome in the intrinsic mitochondrial pathway, initiator caspases are activated through oligomerization resulting in the downstream activation of effector caspases and cleavage of numerous death substrates (Stroh and Schulze-Osthoff, 1998; Fischer et al., 2003).
The HSV-Tk/GCV treatment results in the death not only of the recipient cells (HSV-Tk+) but also of surrounding non-recipient tumor cells. Located between the capsid and the envelope, the tegument is a highly stable macromolecular structure consisting of proteins critical to viral survival (4, 28). pDG, a generous gift from Dr Kleinschmidt (DKFZ, Heidelberg, Germany), is a helper plasmid and provides all genes required for rAAV-2 production in trans. The physicochemical properties that govern these desirable pharmacokinetic characteristics of both prodrugs and effectors are beginning to be understood, and include molecular size, overall lipophilicity, charge, rate of metabolism, and the propensity to form reversible or irreversible complexes with cellular macromolecules. The construct pMRV-CMV-TK/eGFP was produced by replacing the NotI hGFP gene from pTR-UF5 with the NotI-digested TK/eGFP fusion gene fragment from pTK-eGFP.21 For generation of pMRV-EF1-TK/eGFP, the CMV promoter/enhancer sequences from pMRV-CMV-TK/eGFP were excised with KpnI–XbaI, replaced by the EF1 promoter from KpnI–XbaI-digested pTR-UF5-EF1-GFP and the Neor-cassette removed by SalI–SalI digestion and religation of the plasmid backbone. There are potential advantages to each approach. To enhance clarity, the name of the plasmid backbone was omitted (e.g.
As the result of this preferential conversion of prodrugs to drugs, toxic drugs are only produced in tumor cells while having minimal exposure to healthy cells. The human embryonic kidney cell line HEK 293 T and the cervix carcinoma line HeLa-RC23 were grown in DMEM supplemented with 10% fetal calf serum (FCS), penicillin (10 IU/ml) and streptomycin (10 g/ml), and were kind gifts from Dr. Kleinschmidt. The sarcoma cell lines A-204 (Rhabdomyosarcoma24), HS-1 (epitheloid sarcoma,25 HT-1080 (fibrosarcoma26), RD-ES (Ewing sarcoma), SK-N-MC (Askin tumor27) and WSKL-1 (uncharacterized sarcoma), were all obtained from the tumor bank of the German Cancer Research Center (DKFZ, Heidelberg, Germany). These cells were grown in RPMI supplemented with 10% fetal calf serum (FCS), penicillin (10 IU/ml) and streptomycin (10 g/ml). Cells were maintained at 37°C/5% CO2. The culture media, penicillin and streptomycin were all obtained from Life Technologies, whereas FCS was purchased from Sigma (Deisenhofen, Germany).
Note that even though SK-N-MC was originally described as a neuroblastoma, it is now widely regarded as Askin tumor (related to Ewing sarcoma28). rAAV-2 particles were produced by two-plasmid transient transfection as described previously.22 In brief, rAAV-2 particles were released by freeze-thawing (-80°–37°C; three cycles), cell debris was removed by centrifugation and the supernatants were pooled. The rAAV-containing lysate was concentrated and purified by a modified procedure previously described by Tamayose et al.29 Briefly, after pre-equilibration with PBS, the lysate was loaded on a column containing 8 ml sulfonated cellulose matrix beads (Cellufine sulfate, Millipore, Eschborn, Germany). rAAV-2 particles were eluted with 10 mM phosphate buffer (pH 7.2) containing 1.0 M NaCl after washing the column thoroughly with PBS. Modification of the protocol: after the freeze–thawing step, a DNase/RNase and filtration procedure was introduced (modified from Auricchio et al30). To 50 ml lysate, 0.5 mg of each DNase I and RNase A were added and the sample was incubated for 30 minutes at 37°C. After 15 minutes of centrifugation at 2000 g the supernatant was subsequently filtered through a 5 m and a 0.8 m pore size filter (both Millipore) and the procedure was continued as mentioned above.
The eluted fractions, 4 or 8 ml each, were further purified and concentrated by sucrose-cushion centrifugation. The samples were loaded on top of two layers of sucrose, 50 and 30%, respectively, and centrifuged for 5 hours in a Beckman ultracentrifuge (SW40ti swingout-rotor; Beckman, München, Germany) at 250,000 g. The viral pellet was resuspended in 1.5 ml Opti-MEM supplemented with 10 IU/ml penicillin and 10 g/ml streptomycin (both Life Technologies), aliquoted in 250 l portions and stored at -80°C until further use. From each step mentioned above, small aliquots were taken for subsequent titration in order to determine the purification and concentration efficiency of the virus batch.