Efficient gene transfer into T lymphocytes by fiber-modified human adenovirus 5

Abstract

Background: The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors.

Results: Four fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate.

Conclusions: HAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.

Keywords: Adenovirus 5 vector; Gene therapy; Human hematopoietic cells.

Fig. 1

Schematic diagram of the construction of fiber-modified human adenovirus 5 (HAdV-5) vectors. a Construction of the shuttle plasmid carrying human EF1a promoter. b Construction of the backbone plasmid carrying modified fiber gene. c Fiber-modified HAdV-5 vectors.All vectors contained the same human EF1a promoter controlled GFP expression cassette inserted into the E1 region and differently modified fiber genes. CMVp, CMV promoter; C-RGD, RGD4C fused to the C-terminus of HAdV-11p fiber; EF1ap, human EF1a promoter; ES, encapsidation signal; HI-RGD, RGD4C inserted into the HI loop of HAdV-11p fiber Knob; ITR, inverted terminal repeat; Knob11p, knob of HAdV-11p fiber; Knob35, knob of HAdV-35 fiber; MCS, multiple cloning site; pA, SV40 polyA signal; Shaft11p, shaft of HAdV-11p fiber; Shaft35, shaft of HAdV-35 fiber; Tail5, tail of HAdV-5 fiber

Fig. 2

Transduction of hematopoietic cell lines by Ad5-EG and F11p-EG. Cells were infected with adenoviral vectors at MOIs of 100 or 500 vp/cell without virus removal (Ad5-EG and F11p-EG), or viruses were discarded by centrifugation and washing after 6 h’ incubation (Ad5-EG 6 h and F11p-EG 6 h). GFP expression was analyzed with flow cytometry assay 2 days post infecton. The percentage of GFP+ cells (a) and the mean fluorescence intensity of GFP+ cells (b) were compared among different vectors and cell lines

Fig. 3

Transduction of hematopoietic cell lines by fiber-modified HAdV-5 vectors. Cells were infected with adenoviral vectors at MOIs of 100 or 500 vp/cell, and GFP expression was analyzed with flow cytometry 2 days post infecton. The percentage of GFP+ cells (a) and the mean fluorescence intensity of GFP+ cells (b) were compared among different vectors and cell lines

Fig. 4

Transduction of cord blood CD34+ cells by fiber-modified HAdV-5 vectors. Isolated cord blood CD34+ cells were infected by adenoviral vectors at an MOI of 1000 vp/cell. Two days post infection, cells were labelled with APC-conjugated anti-CD34 antibody, and the GFP and APC fluorescences were analyzed with flow cytometry

Fig. 5

Transduction of primary human T lymphocytes by fiber-modified HAdV-5 vectors. T cells were isolated from the peripheral blood of heath donors, expanded through activation with anti-CD3 and anti-CD28 antibodies coated beads, infected by adenoviral vectors at MOIs of 100 or 500 vp/cell. GFP expression was analyzed with flow cytometry 2 days post infection. The results of the percentage of GFP+ cells and the mean fluorescence intensity of GFP+ cells were shown

Fig. 6

Transduction of CD4+ or CD8+ T cells by fiber-modified HAdV-5 vectors. T cells were isolated, expanded and infected by adenoviral vectors at MOIs of 500 or 1000 vp/cell. Two days post infection, cells were labelled with fluorescein-conjugated anti-CD3, anti-CD4 and anti-CD8 antibodies. GFP and fluoresceins were analyzed with flow cytometry. The data processing procedure was shown (a). Live lyphocytes were gated and separated from the cellular debris on the FSC-H vs. SSC-H dot plot, and CD3+ T cells were then gated and grouped according to the expression of CD4 or CD8 molecules. GFP expression in CD4 + CD8- or CD4-CD8+ subgroups were separately analyzed (b), and the percentages of CD4 + CD8- or CD4-CD8+ cells in CD3+ T cells were calculated (c)

Fig. 7

Effect of viral infection on the ratio of CD4/CD8 T cells. T cells were isolated, expanded and infected by adenoviral vectors at an MOI of 1000 vp/cell. At 0, 24, 48 and 72 h post infection (hpi), cells were harvested and labelled with fluorescein-conjugated anti-CD4 and anti-CD8 antibodies. GFP and fluoresceins were analyzed with flow cytometry. The percentages of CD4 + CD8- (a) or CD4-CD8+ (b) T cells were calculated and sequentially displayed according to the order of culture time. The data of the uninfected group served as a control. The percentage of GFP+ cells in CD4+ or CD8+ T cell subsets was displayed to show the dynamic expression of GFP (c)

Fig. 8

Effect of viral infection on the growth of T cells. T cells were isolated, expanded, stained with Dye eFluor 670, and infected by adenoviral vectors at an MOI of 1000 vp/cell. At 0, 24, 48 and 72 h post infection (hpi), cells were harvested and fixed in 1.5% paraformaldehyde in PBS. Fluorescence of GFP and eFluor 670 were analyzed with flow cytometry at the end of the experiment. Proliferation index (PI) of the total cells were calculated (a), or PI values of GFP- or GFP+ cells were separately calculated (b)