Figure 6. Minimal mobilization of ECOO comprising SIN vectors.

Mobilization efficiency of the SIN ECOO-comprising vector pTK1940 and its conventional SIN and non-SIN counterparts vectors, pTK945 and pTK1087, respectively. (A) Depiction of conventional and ECOO-comprising non-SIN/SIN vectors (pTK1087/pTK945 and pTK1976/pTk1940, respectively). The non-SIN U3 in the 3’LTR’s are indicated. Arrows indicate the direction of transcription from the internal CMV promoter and the orientation of the WPRE, SV40 and BGH poly-A signals. (B) Lentiviral vector mobilization from episomal vector genomes at 72h post IDLV transduction of the tetracycline regulated stable packaging cell line, SODk1, either in the presence of doxycycline or in the absence of doxycycline and the presence of sodium butyrate (uninduced or, induced respectively). Mobilized vector particles were employed on naïve 293T target cells. To dilute out carry over of non-mobilized IDLV’s, titers of mobilized ICLV’s (generated by the induced packaging cells) were determined following 5 passages of the above target cells in culture. Percentage of GFP-positive cells and the number target cells at the time of transduction were used to calculate vector titers. Black and white bars describe titers (TU/ml) of the above vectors mobilized from induced and uninduced SODk1 cells, respectively. Error bars represent standard deviation of 3 independent experiments. (C) Lentiviral vector mobilization from integrated vector genomes (pTK1087, pTK945 and pTK1940) in induced and uninduced SODk1 cells. Conditioned media were employed on 293T cells and vector titers were determined by scoring GFP positive cells following serial dilutions on 293T cells. Black and white bars describe titers (TU/ml) of the above vectors mobilized from induced and uninduced SODk1 cells, respectively. The results are average of three independent experiments. Asterisk indicates titers lower than detection level (<10TU/mL). (D) Normalized vector titers. To minimize bias of VCN in SODk1 on mobilized vector titers, calculated mobilization titers from ICLV-transduced SODk1 cells were normalized to VCN (vector producing SODk1 cells) as determined by real time PCR. Black and white bars describe normalized titers (TU/ml/copy) of the above vectors mobilized from induced and uninduced SODk1 cells, respectively. Titers were represented as average ± SD of 3 independent experiments. (E-F) Characterizing the combined effect of SIN-LTRs and the orientation of lentiviral vector internal expression cassettes on mobilization of integrated lentiviral vectors. PKR-deficient 293T cells transduced with integrating ECOO-comprising non-SIN vector (pTK1976) and naïve 293T cells transduced with either, conventional non-SIN and SIN vectors (pTK1087 and pTK945, respectively) or ECOO-comprising SIN and non-SIN vectors (pTK1976 and pTK1940, respectively) were analyzed for vector copy number by qPCR. The aforementioned vector-transduced 293T cell were transfected with a VSV-G envelope and the ΔNRF packaging cassettes. Mobilized vectors in conditioned media were collected at 72 hours post-transfection and tittered by scoring GFP expression following serial dilutions on 293T cells. (E) Bar graph describing titers of mobilized lentiviral vectors from integrated vector genomes in naïve (black bars) and PKR deficient (white bars) 293T cells. The results are average of three independent experiments. (F) Bar graph showing the above titers of mobilized vectors after normalization for VCN. The results are average of three independent experiments.