S. Although titers of retroviral vectors were not clearly influenced by
S. Although titers of retroviral vectors were not clearly influenced by insertion of single-copy of the mutant tRNALys3 genes, the retroviral constructs carrying multiple copies of the Mt13TD gene showed an apparent pattern of significant decrease in titers (p < 0.001) (Figure 4A). The vector titer from the triple copies of Mt13TD construct dropped from 3.03 ?0.25 ?106 IU/mL to 2.25 ?0.35 ?105 IU/mL compared with that of the construct carrying single-copy of Mt13TD?a 13-fold decrease, which was also seen where the titer derived from the construct with 12 copies of the Mt13TD gene dropped to 3.72 ?0.12 ?103 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28878015 IU/mL. To overcome the challenge due to decreased titers from these vector constructs, clones of transduced cells were obtained through a limiting-dilution method and tested for viral inhibition with replication competent HIV-1. In spite of marked Pyrvinium embonate web variations among different clones, Figure 4B shows that cells transduced with the construct carrying 3 copies of Mt13TD showed lower TCID50 titers compared to cells transduced with single-copy of the gene. Consistently, cells transduced with the construct carrying 6 copies of the gene generally showed lower TCID50 titers than cells transduced with 3 copies. Furthermore, cells transduced with the construct carrying 12 copies showed the lowest TCID50 titers, with exception of two clones. Based on the results from the TCID50 test, two of the clones transduced with the 12-copy construct were further evaluated by challenging with HIV-1 at MOIs of 0.1 and 1.0 respectively. As shown in Figures 4C and 4D, these two clones showed significant reduction of HIV-1 replication than cells transduced with a single copy of the same gene or the wild-type tRNALys3, especially the latter (p < 0.001). When challenged at MOI 1.0, peak production of P24 from cells transduced with the wild-type tRNALys3 occurred at day 6 pi with massive cell death, and P24 production decreased sharply following that time point. When these control cells were infected at MOI 0.1, the peak production of P24 occurred on day 12 pi, with the absolute concentration of the peak level 1.26 times higher than that of the cellsWu et al. Retrovirology 2013, 10:112 http://www.retrovirology.com/content/10/1/Page 6 ofFigure 4 Characterization of CEM-SS cells transduced with retroviral constructs carrying multiple copies of the Mt13TD gene. (A) Comparison of titers of vector preparations. (B) TCID50 tests on cloned cells. Numbers of 3, 6, 9 and 12 correspond to copies of the gene in the retroviral constructs used to transduce the cells. NT, non-transduced. (C) Based on results in (B), two clones of cells transduced with Mt13TD-12, namely No. 3 and No. 5 respectively, were challenged with HIV-1 at MOI of 1.0. Cells transduced with vectors containing one copy of either the wild-type tRNALys3 or the Mt13TD gene were challenged and sampled simultaneously as controls. (D) The same type of cells as in (C) were challenged at a lower MOI of 0.1 and sampled.infected at MOI 1.0. In contrast, when challenged at MOI 1.0, the cells transduced with the vector carrying singlecopy of Mt13TD accumulated concentration of P24 2 logs lower with occurrence of the peak level delayed to day 9 pi. Following that time point, the P24 level remained relatively stable with a slight decrease. Similarly, when challenged at PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26795252 a lower MOI of 0.1, the P24 accumulated slower with concentrations greater than 2 logs lower compared with that of the cells transduced with the wild-type tR.
