Error bars indicate the standard deviation. post-infection, cells were analyzed for EGFP expression via circulation cytometry. EGFP expression is usually shown in IM-9, Z-138, and REC-1 human cell lines with VSV-G, Polidocanol 2.2 SINDBIS and BAP SINDBIS. Representative data are shown. See Furniture 2C4 for summary of impartial trials. Supplementary Physique 4. Representative dot plots of EGFP expression in T cells. Cells were infected with computer virus conjugated to TfR1 targeting antibodies for 2 hours. Four days post-infection, cells were analyzed for EGFP expression via circulation cytometry. EGFP expression is usually shown in Jurkat and MOLT-4 human cell lines with VSV-G, 2.2 SINDBIS and BAP SINDBIS. Representative data are shown. See Furniture 2C4 for summary of impartial trials. Supplementary Physique 5. Delivery of FCU1 into MM.1S cells and the induction of cell death in the presence of 5-FC. MM.1S cells were infected with the indicated computer virus particles with VSV-G envelope, 2.2 SINDBIS envelope conjugated with ch128.1, Polidocanol and BAP SINDBIS envelope T conjugated with ch128.1Av. BAP SINDBIS viruses where also conjugated to IgG3-Av as a negative, non-targeting moiety. Two-hours post-transductions cells were treated with numerous concentrations of 5-FC for 4 days. Direct treatment with 0.1mg/ml 5-FU was used as positive control. Cell viability was measured using the MTS assay. Data are the averages of 3 impartial experiments and data are offered as a percentage of cells transduced with the same computer virus in the absence of 5-FC. Error bars indicate the standard deviation. indicates < 0.05 and shows significant difference when compared to control cells transduced but without the addition of 5-FC (unless indicated otherwise). NIHMS581748-supplement-Supplementary_Figurs.pdf (6.5M) GUID:?D86973E0-3A3B-479D-95CD-9EA6F9D28473 Abstract Background We previously designed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for malignancy therapy and showed that ch128.1Av delivers the biotinylated herb toxin saporin-6 into malignant B cells. However, due to common expression of TfR1, delivery of the toxin to normal cells is usually a concern. Therefore, we explored the potential of dual targeted lentiviral-mediated gene therapy approaches to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. Methods Circulation cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene models of MM [33]. Conjugation of ch128.1Av with biotinylated saporin 6, a herb ribosomal inactivating toxin, overcame resistance of malignant B-cells to the treatment of ch128.1Av [34]. The mechanism of cell death induced by ch128.1Av conjugated to this toxin was shown to be due to the effects of the toxin and not iron starvation [35], suggesting the ability of ch128.1Av to deliver active anti-cancer agents into TfR1 overexpressing malignant cells. ch128.1Av alone is not toxic to normal hematopoietic stem/early progenitor cells [33] or late progenitors [34]. However, conjugation of ch128.1Av with biotinylated saporin was highly toxic to late progenitor cells of both the erythroid and myeloid lineages [34]. Importantly, no toxicity to hematopoietic stem/early progenitor cells was observed upon treatment with the ch128.1Av complexed with biotinylated saporin [35], which is consistent with the lack of TfR1 expression on these cells [36C38]. To overcome the potential side effects of the delivery of harmful proteins into normal cells expressing the TfR1, we have developed a new gene therapy strategy. We have previously shown targeted delivery of enhanced green fluorescent protein (EGFP) into Jurkat T cell leukemia cells using biotinylated lentiviral vectors conjugated to ch128.1Av [39]. Lentiviruses were chosen since they can transduce non-dividing cells and are less immunogenic than their adenoviral counterparts [40]. The Polidocanol goal of the current study was to expand that approach and develop dual targeted strategies using targeted lentiviral-mediated gene delivery for the treatment of B cell malignancies. Since the TfR1 is usually overexpressed on the surface of malignancy cells and in order to enhance the therapeutic window, the first level of targeting occurs through the use of our antibodies specific for the TfR1 in two different strategies: 1) lentiviral vectors pseudotyped with a altered chimeric Sindbis computer virus envelope encoding the Fc-binding region (ZZ domain name) of protein A (2.2 SINDBIS) conjugated to ch128.1, and 2) pseudotyped computer virus containing the biotin adaptor peptide (BAP SINDBIS) instead of the ZZ domain name, which results in biotinylation of the Polidocanol surface of the computer virus, conjugated to ch128.1Av. The second level of targeting will occur through the.