Volume 14, Issue 4, Pages 555-563 (October 2006) Construction and Use of Retroviral Vectors Encoding the Toxic Gene Barnase  Sumit Agarwal, Bryan Nikolai, Tomoyuki Yamaguchi, Patrycja Lech, Nikunj V. Somia  Molecular Therapy  Volume 14, Issue 4, Pages 555-563 (October 2006) DOI: 10.1016/j.ymthe.2006.03.025 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Illustration of vectors used in this study. (A) CSII-EF-Barnase. Lentiviral vector for transduction of the barnase gene. The vector is a self-inactivating vector, with a deletion in the 3′ U3 LTR (ΔU3). RRE, Rev-responsive element; cPPT, central polypurine tract; EF-1α, elongation factor 1α promoter; PRE, posttranscriptional regulatory element; SD/SA, splice donor and acceptor sequences. Genes for the enhanced green fluorescent protein (EGFP), blasticidin resistance (Bsd), and luciferase (Luc) were also expressed from this vector. (B) pCLMFG-barnase. MLV-based vector transducing barnase. The vector expresses barnase in infected cells regulated by the MLV LTR. EGFP was also cloned into the pCLMFG vector. (C) pRK5-barstar. Mammalian expression of the barnase inhibitor barstar is directed by the CMV promoter. pA is the SV40 polyadenylation signal. EGFP was also expressed from this vector to monitor transfection efficiency. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Transfection of barnase and barstar in 293T cells. MLV barnase vectors were generated by transient transfection of 293T cells as outlined under Material and Methods (A and B) with or (C and D) without barstar. An expression plasmid for EGFP was added to verify transfection efficiency and expression was visualized by fluorescence microscopy (B and D). A and C are bright-field images of B and D. Note that the image capture time for B was 400 ms, while it was increased to 800 ms for D to allow visualization of the low EGFP expression. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Barnase transduction in mammalian cells. (A) Cell killing and dose response after infection with a MLV-based barnase vector. (Graph A) A human cervical adenocarcinoma cell line, HeLa. (Graph B) A human prostate cancer cell line, DU-145. (Graph C) A human kidney carcinoma cell line, A-498. (Graph D) A hamster lung fibroblast cell line. Cells were infected with twofold dilutions of the barnase vector generated in the presence (square) or the absence (diamond) of barstar. Results are expressed as percentage cell survival of uninfected cells. Assays were conducted in quadruplicate for each point, and the means and plusmn; SEM are shown. (B) Cell killing and dose response after infection with a lentiviral-based barnase vector. Cell lines, conditions, and symbols are identical to those of the MLV experiment. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Transduction and growth rate of 31-2 cells. (A) HIV-1-based vector particles were generated by transient transfection of vector plasmid transducing the blasticidin resistance gene. The viral vector was used to infect V79-4 and 31-2 cells. The HIV-1 vector DNA was also transfected into V79-4 and 31-2 cells. Gene transfer was assayed by the number of blasticidin-resistant colonies and the data are presented as the relative number of colonies compared to the wild-type cells. (B) The growth rates of V79-4 and 31-2 cells were compared over a 108-h period. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Transduction of 31-2 cells. (A) Dose response to luciferase transduction. HIV-1-based vectors transducing luciferase were serially diluted 10-fold and used to infect V79-4 and 31-2 cells. Data are shown as the percentage infection relative to wild-type cells within each pair of experimental samples at the same m.o.i. (B) Relative cell infection by lenti- and MLV vectors. MLV and HIV-1 vector preparations transducing EGFP were used to infect V79-4 and 31-2 cells at an m.o.i. of 1 (assayed on HeLa cells). The average of the mean fluorescence intensity (for HIV-1 vectors n = 4) was 763 for V79-4 cells and 569 for 31-2 cells. The graph shows representative data from infections assayed by flow cytometry. (C) Infection mediated by MLV amphotropic envelope. HIV-1-based vectors transducing luciferase pseudotyped with the MLV 10A1 amphotropic envelope were used to infect V79-4 and 31-2 cells. Data are shown as the percentage infection relative to wild-type cells. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 qPCR assay to follow the progress of reverse transcription (RT) in V79-4 and 31-2 cells. (A) CSII-EGFP vector RNA is shown with the relative positions of the primers used for the qPCR analysis. Primer pairs in the LTR9 and AA55 detect the negative-strand strong-stop cDNA; U31 and U32 detect products after the first jump and 5NC and LTR9 allow products to be detected at the end of the process. (B) Quantification of strong stop (SS), first jump (1st J), and late products (full) over time after infection of V79-4 cells. The amount of product is expressed relative to the amount of β-actin transcript (ΔCT). Controls are no virus added and addition of heat-inactivated viral vector (HIA). A decrease in the ΔCT value reflects an increase in the amount of viral DNA. (C) Quantification of RT products over time in 31-2 cells. Notation is identical to that of infection of V79-4 cells. Molecular Therapy 2006 14, 555-563DOI: (10.1016/j.ymthe.2006.03.025) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions